JPWO2007142277A1 - Monoclonal antibodies that bind to heparin-binding epidermal growth factor-like growth factor. - Google Patents

Abstract

There is a need for a medicament for treating diseases in which HB-EGF is enhanced. According to the present invention, it is possible to provide monoclonal antibodies and antibody fragments thereof that bind to HB-EGF, membrane-type HB-EGF, and secretory HB-EGF bound to the cell membrane.

Description

  The present invention relates to heparin-binding epidermal growth factor-like growth factor (hereinafter referred to as HB-EGF), membrane-type HB-EGF, and secretory-type HB-EGF. The present invention relates to monoclonal antibodies that bind to and antibody fragments thereof.

  HB-EGF was purified and isolated from the culture supernatant of human macrophage-like cell line U-937 differentiated into macrophages by Higashiyama et al. In 1992 (Non-patent Document 1). HB-EGF shares the six cysteines conserved in the epidermal growth factor (EGF) family, belongs to the EGF family, and, like other proteins belonging to the EGF family, type I It is synthesized as a membrane protein (Non-patent Documents 1 and 2). Membrane-type HB-EGF was activated by various physiological stimuli such as heat and osmotic stress, growth factors, cytokines, and lysophosphatidic acid (LPA), a G protein-coupled receptor (GPCR) agonist It is converted into secretory HB-EGF of 14 to 22 kilodalton (hereinafter referred to as kDa) by metalloprotease (Non-patent Documents 1 to 3). Secreted HB-EGF binds to EGF receptor (EGFR / ErbB1) (Non-patent document 1), ErbB4 (Non-patent document 4) and N-arginine dibasic convertase (Non-patent document 5), and fibroblasts And smooth muscle cells (Non-patent document 1), keratinocytes (Non-patent document 6), hepatocytes (Non-patent document 7), and mesangial cells (Non-patent document 8). In addition, organ formation such as heart valves (Non-Patent Documents 28, 29 and 31), wound healing (Non-Patent Documents 9 and 10), smooth muscle cell hyperplasia caused by atherosclerosis (Non-Patent Document 11), Restenosis (Non-patent documents 12 and 13), Pulmonary hypertension (Non-patent document 14), Liver regeneration (Non-patent document 15), Brain disorders (Non-patent document 16) and Cancer (Non-patent documents 28 to 35) It is also known that HB-EGF is involved.

  On the other hand, it has been reported that a considerable amount of membrane-type HB-EGF is expressed on the cell surface without being cleaved into a secreted form (Non-patent Document 17). Membrane-type HB-EGF is known to form a complex with tetraspanins such as CD9 and integrin α3β1 on the cell surface, and there is a report that it interacts with neighboring cells as a jack-cline growth factor. (Non-patent documents 17 to 22). Naglich et al. Have reported that membrane-type HB-EGF functions as a receptor for diphtheria toxin and is involved in the entry of diphtheria toxin into cells (Non-patent Document 23).

Mekada et al. Created HB-EGF knockout (KO) mice and analyzed the physiological functions of HB-EGF. As a result, HB-EGF KO mice showed symptoms of ventricular dilatation, decreased cardiac function, and heart valve hypertrophy. More than half died within a few days after birth. This indicates that HB-EGF is an essential protein for cardiac development and functional maintenance (Non-patent Document 24).
Next, Mikada et al. Secreted HB-EGF (hereinafter referred to as HB ^uc ) that is no longer converted into a secreted form by introducing a mutation at the protease cleavage site, and a protease-independent secretion that lacks the transmembrane region. Two types of genes of HB-EGF (hereinafter referred to as HB ^Δtm ) were prepared. Recombinant mice expressing the respective HB-EGF mutants were prepared, and the physiological functions of membrane-type and secreted HB-EGF were analyzed (Non-patent Document 25). As a result, HB ^uc- expressing mice showed symptoms similar to HB-EGF KO mice, suggesting that secreted HB-EGF functions as an active protein. Most HB ^Δtm expressing mice died before or during the neonatal period. Furthermore, keratinocyte hyperplasia and ventricular hypertrophy from the neonatal period were observed in HB ^{Δtm / +} mice in which mutation was introduced into only one of the alleles. These symptoms were the opposite phenotype of HB-EGF KO mice and HB ^uc expressing mice. CRM197 (non-patent document 26) known as a diphtheria toxin mutant specifically inhibits the cell growth promoting activity of HB-EGF and does not permeate the cell membrane. The CRM197 is, since the inhibited hyperplasia and ventricular hypertrophy is a phenotype of HB ^{△ tm} expressing mice, HB ^{△ tm} HB ^{△ tm} produced by expressing mice can bind to receptors in the cell prior secretion Rather than acting, it is presumed to act by binding to cell surface receptors after being secreted extracellularly. Therefore, the quantitative balance between membrane-type HB-EGF and secretory HB-EGF in vivo is essential for maintaining normal physiological functions, and conversion of membrane-type HB-EGF from secretory to secretory type in vivo The process is considered controlled.

Higashiyama et al. Found that secreted HB-EGF protein in the heart increased in mice with stenosis of the thoracic aorta to induce cardiac hypertrophy. When these mice were administered a low molecular weight compound that inhibits a protease that converts membrane-type HB-EGF to secretory form, the conversion of membrane-type HB-EGF to secretory form in the heart was suppressed, resulting in suppression of cardiac hypertrophy. (Non-patent document 27).
So far, it has been reported that HB-EGF is highly expressed in various cancers such as breast cancer, liver cancer, pancreatic cancer, and bladder cancer as compared with normal tissues (Non-Patent Documents 28 to 31). Recently, it has been clarified that HB-EGF is an important factor for cancer growth (Non-patent Documents 32 and 33). In a model system for transplanting human ovarian cancer cell lines into nude mice, Mekada et al. Introduced HB-EGF small interference RNA (siRNA) into cancer cell lines, or administered CRM197 to mice transplanted with cancer cell lines As a result, it was revealed that a remarkable tumor growth inhibitory effect was observed. Higashiyama et al. Also introduced the HB-EGF gene into a bladder cancer cell line, and increased cell proliferation, colony-forming ability, vascular endothelial growth factor (VEGF) expression, and cyclin D1 expression in vitro. Was revealed. In vivo, it was also reported that increased tumorigenicity and tumor angiogenesis were observed in nude mice. Such growth-promoting action is observed only when the membrane-type HB-EGF or secretory HB-EGF gene is expressed, and is observed when the protease-resistant membrane-type HB-EGF gene is forcibly expressed. There wasn't. Therefore, it was suggested that secreted HB-EGF may be an important factor related to tumor growth of ovarian cancer and bladder cancer. As for HB-EGF expression in clinical patients, Mekada et al. Analyzed HB-EGF mRNA expression level in tumor tissues of ovarian cancer patients and secreted HB-EGF protein concentration in ascites. It has been reported that only -EGF has increased expression (Non-patent Document 32). Furthermore, Miyamoto et al. Reported that the prognosis of ovarian cancer patients with high expression of tumor HB-EGF mRNA was poorer than that of patients with low expression (Non-patent Document 34). The above results indicate that, at least in ovarian cancer, secreted HB-EGF produced by cancer is involved in cancer growth by the autocrine or paracrine mechanism (Non-patent Document 35). As antibodies that inhibit the activity by binding to secretory HB-EGF, several polyclonal antibodies and one kind of monoclonal antibody (both manufactured by R & D) are known. It has been reported that anti-HB-EGF goat polyclonal antibody (manufactured by R & D) binds to cell surface membrane-type HB-EGF expressed in COS-7 cells (Non-patent Document 3). It is widely known that when a membrane protein is present on the surface of a cell such as cancer, a monoclonal antibody that binds to the protein can be a therapeutic agent that inhibits the proliferation of the cell (Non-patent Document 36). However, secretory HB-EGF, HB-EGF bound to the cell membrane, and monoclonal antibody that binds to membrane HB-EGF have not been reported so far.
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    There is a need for a medicament for treating diseases involving HB-EGF.

The present invention relates to the following (1) to (24).
(1) Heparin binding epidermal growth factor-like growth factor (hereinafter referred to as HB-EGF), membrane-type HB-EGF and secretory HB-EGF A monoclonal antibody or antibody fragment thereof that binds.
(2) The monoclonal antibody or antibody fragment thereof according to (1), which binds to an epidermal growth factor-like domain (EGF-like domain) of HB-EGF, membrane-type HB-EGF and secretory HB-EGF bound to a cell membrane.
(3) The monoclonal antibody or antibody fragment thereof according to claim 1 or 2, which inhibits binding between secretory HB-EGF and HB-EGF receptor.
(4) The monoclonal antibody or antibody fragment thereof according to any one of (1) to (3), which has neutralizing activity against secretory HB-EGF.
(5) The monoclonal antibody or the antibody fragment thereof according to any one of (1) to (4), which binds to a binding region between secretory HB-EGF and HB-EGF receptor or diphtheria toxin.
(6) The monoclonal antibody or the antibody thereof according to (1) to (5), which binds to an epitope containing at least one amino acid among the 133rd, 135th, and 147th amino acid sequences represented by SEQ ID NO: 2 fragment.
(7) The monoclonal antibody or the antibody fragment thereof according to (6), which binds to an epitope comprising amino acids 133, 135 and 147 of the amino acid sequence represented by SEQ ID NO: 2.
(8) The monoclonal antibody or antibody fragment thereof according to (1) to (5), which binds to an epitope comprising the 141st amino acid of the amino acid sequence represented by SEQ ID NO: 2.
(9) The monoclonal antibody or antibody fragment thereof according to (1) to (3), (5) and (8), which binds to an epitope to which a monoclonal antibody produced by hybridoma KM3579 (FERM BP-10491) binds.
(10) The monoclonal antibody or antibody fragment thereof according to any one of (1) to (7), which binds to an epitope to which a monoclonal antibody produced by hybridoma KM3567 (FERM BP-10573) binds.
(11) The monoclonal antibody or the antibody fragment thereof according to any one of (1) to (7), which binds to an epitope to which a monoclonal antibody produced by hybridoma KM3566 (FERM BP-10490) binds.
(12) Complementarity determining region (hereinafter referred to as CDR) 1, CDR2 and CDR3 of antibody heavy chain variable region (hereinafter referred to as VH) are represented by SEQ ID NOs: 12, 13 and 14, respectively. The CDR1, CDR2, and CDR3 of the light chain variable region (hereinafter referred to as VL) of an antibody each include an amino acid sequence represented by SEQ ID NOs: 15, 16, and 17, respectively (1) to (1) The monoclonal antibody or antibody fragment thereof according to any one of 7) and (11).
(13) The antibody or antibody fragment thereof according to any one of (1) to (12), wherein the monoclonal antibody is a gene recombinant antibody.
(14) The antibody or antibody fragment thereof according to (13), wherein the recombinant antibody is selected from a human chimeric antibody, a humanized antibody and a human antibody.
(15) The human chimeric antibody according to (14), wherein VH of the human chimeric antibody includes the amino acid sequence represented by SEQ ID NO: 9, and VL includes the amino acid sequence represented by (11). Or an antibody fragment thereof.
(16) The VH of the humanized antibody is the amino acid sequence represented by SEQ ID NO: 22 or the 9th Ala of the amino acid sequence represented by SEQ ID NO: 22 to Thr, the 20th Val to Leu, and the 30th Thr To Arg, 38th Arg to Lys, 41st Pro to Thr, 48th Met to Ile, 67th Arg to Lys, 68th Val to Ala, 70th Ile to Leu The amino acid sequence introduced with at least one modification selected from the modification that replaces 95th Tyr with Phe and 118th Val with Leu, and
The VL of the humanized antibody has the amino acid sequence represented by SEQ ID NO: 23 or the 15th Leu of the amino acid sequence represented by SEQ ID NO: 23 as Val, the 19th Ala as Val, and the 21st Ile as Met. The humanized antibody or antibody fragment thereof according to (14), comprising an amino acid sequence into which at least one modification selected from a modification in which 49th Pro is replaced with Ser and 84th Leu is replaced with Val is introduced.
(17) A peptide in which the antibody fragment comprises Fab, Fab ′, F (ab ′) ₂ , single chain antibody (scFv), dimerization V region (Diabody), disulfide stabilized V region (dsFv) and CDR The antibody fragment according to any one of (1) to (16), which is an antibody fragment selected from:
(18) A DNA encoding the antibody or antibody fragment thereof according to any one of (1) to (17).
(19) A recombinant vector containing the DNA according to (18).
(20) A transformant obtained by introducing the recombinant vector according to (19) into a host cell.
(21) The transformant according to (20) is cultured in a medium, and the antibody or antibody fragment thereof according to any one of (1) to (17) is produced and accumulated in the culture, The method for producing an antibody or antibody fragment thereof according to any one of (1) to (17), wherein the antibody or the antibody fragment is collected.
(22) A medicament comprising the antibody or antibody fragment thereof according to any one of (1) to (17) as an active ingredient.
(23) A therapeutic agent for a disease involving HB-EGF, comprising the antibody or antibody fragment thereof according to any one of (1) to (17) as an active ingredient.
(24) The therapeutic agent according to (23), wherein the disease involving HB-EGF is cancer.

  According to the present invention, it is possible to provide an antibody that binds to HB-EGF, membrane-type HB-EGF, and secretory HB-EGF bound to the cell membrane.

The reactivity of various anti-HB-EGF monoclonal antibodies in binding ELISA is shown. The horizontal axis represents the concentration of each antibody, and the vertical axis represents the binding activity of each antibody. ◇ represents the monoclonal antibody KM511, ■ represents the monoclonal antibody KM3566, Δ represents the monoclonal antibody KM3567, ▲ represents the monoclonal antibody KM3579, and ◯ represents the monoclonal antibody MAB259. The anti-HB-EGF monoclonal antibodies KM3566, KM3567, KM3579 and MAB259 show the HB-EGF-EGFR binding inhibitory activity. The horizontal axis shows the concentration of each antibody, and the vertical axis shows the binding of biotin-labeled HB-EGF in fluorescence intensity. The horizontal solid line shows the fluorescence intensity when biotin-labeled HB-EGF was added and when no antibody was added, and the horizontal dotted line shows the fluorescence intensity when biotin-labeled HB-EGF was not added and no antibody was added. Δ represents monoclonal antibody KM3566, × represents monoclonal antibody KM3567, ● represents monoclonal antibody KM3579, and ■ represents monoclonal antibody MAB259. The HB-EGF neutralizing activity of various anti-HB-EGF monoclonal antibodies is shown. The horizontal axis represents the concentration of each antibody, and the vertical axis represents the growth inhibition rate (%). ◇ represents monoclonal antibody KM511, ■ represents monoclonal antibody KM3566, ▲ represents monoclonal antibody KM3579, and ◯ represents monoclonal antibody MAB259. The HB-EGF neutralizing activity of various anti-HB-EGF monoclonal antibodies is shown. The horizontal axis represents the concentration of each antibody, and the vertical axis represents cell proliferation. HB-EGF (+) indicates cell growth when HB-EGF is added and without antibody, and HB-EGF (−) indicates cell growth when HB-EGF is not added and when no antibody is added. □ represents monoclonal antibody MAB259, ■ represents monoclonal antibody KM3567, and ▲ represents monoclonal antibody KM3566. The reactivity of various anti-HB-EGF monoclonal antibodies in FCM analysis is shown. The horizontal axis represents the concentration of each antibody, and the vertical axis represents the average fluorescence intensity MFI value. X represents the monoclonal antibody KM511, Δ represents the monoclonal antibody KM3566, □ represents the monoclonal antibody KM3579, and ◯ represents the monoclonal antibody MAB259. The reactivity of various anti-HB-EGF monoclonal antibodies with respect to MDA-MB-231 cells in FCM analysis is shown. The solid line in each histogram indicates the negative control antibody KM511, and the broken line indicates each anti-HB-EGF antibody. (A) MAB529, (b) KM3566, (c) KM3567, (d) KM3579 are shown. The construction process of the anti-HB-EGF chimeric antibody expression vector pKANTEX3566 is shown. The electrophoresis pattern of SDS-PAGE (5-20% gradient gel use) of purified anti-HB-EGF chimeric antibody KM3966 is shown. Lane 1 shows the molecular weight marker, Lane 2 shows the anti-HB-EGF chimeric antibody KM3966 under reducing conditions, and Lane 3 shows non-reducing conditions. The reactivity of the anti- HB-EGF chimeric antibody KM3966 with respect to a human solid cancer cell line in flow cytometry is shown. In the figure, the vertical axis represents the number of cells, and the horizontal axis represents the fluorescence intensity. The reactivity of anti-HB-EGF chimeric antibody KM3966 to human solid cancer cell lines treated with recombinant HB-EGF in flow cytometry is shown. In the figure, the vertical axis represents the number of cells, and the horizontal axis represents the fluorescence intensity. The neutralizing activity with respect to human HB-EGF of the anti-HB-EGF chimeric antibody KM3966 is shown. In the figure, the vertical axis represents the absorbance value of O.D450 nm representing the number of living cells, and the horizontal axis represents the antibody concentration. ■ indicates negative control antibody human IgG, and □ indicates KM3966. HB-EGF (-) indicates no addition of HB-EGF, and HB-EGF (+) indicates addition of HB-EGF. 2 shows antibody-dependent cytotoxic activity (ADCC activity) of an anti-HB-EGF chimeric antibody KM3966 against a human solid cancer cell line. In the figure, the vertical axis represents the cytotoxic activity rate (%), and the horizontal axis represents the antibody concentration of the anti-HB-EGF chimeric antibody KM3966. The horizontal line shows the cytotoxic activity when no antibody was added. The anti-tumor activity in the early cancer model of anti-HB-EGF chimeric antibody KM3966 is shown. The vertical axis in the figure represents the tumor volume, and the horizontal axis represents the number of days after cancer cell transplantation. ● represents the PBS administration group, and ○ represents the KM3966 10 mg / kg administration group. Bars indicate standard deviation. The anti-tumor activity in the advanced cancer model of anti-HB-EGF chimeric antibody KM3966 is shown. The vertical axis in the figure represents the tumor volume, and the horizontal axis represents the number of days after cancer cell transplantation. ● represents the PBS administration group, and ○ represents the KM3966 10 mg / kg administration group. Bars indicate standard deviation. The reactivity with the anti-HB-EGF mouse antibody KM3566 with respect to the human hematological cancer cell line | wire by flow cytometry is shown. In the figure, the vertical axis represents the number of cells, and the horizontal axis represents the fluorescence intensity. A represents an acute myeloid leukemia cell line, and B represents a T cell leukemia cell line. 2 shows antibody-dependent cytotoxic activity (ADCC activity) of anti-HB-EGF chimeric antibody KM3966 against human blood cancer cell lines. In the figure, the vertical axis represents the cytotoxic activity rate (%), and the horizontal axis represents the antibody concentration of the anti-HB-EGF chimeric antibody KM3966. The horizontal line shows the cytotoxic activity when no antibody was added. The reactivity of anti-HB-EGF monoclonal antibodies KM3566 and KM3579 and chimeric antibody KM3966 to mutant HB-EGF-expressing cells is shown. In the figure, the vertical axis represents the reactivity (%) of each antibody, and the horizontal axis represents the type of mutant HB-EGF.

  In the present invention, the membrane type HB-EGF has a transmembrane domain and binds to the cell membrane, and is composed of a signal sequence, a pro region, a heparin binding domain, an EGF-like domain, a Jackson membrane domain, and a cytoplasmic domain. HB-EGF. Specific examples include a polypeptide having the amino acid sequence represented by SEQ ID NO: 2. In the present invention, secretory HB-EGF refers to an extracellular domain containing an EGF-like domain in which the membrane-binding site of membrane-type HB-EGF has been cleaved with a protease or the like. Specific examples include a polypeptide having the amino acid sequence represented by SEQ ID NO: 3. HB-EGF bound to the cell membrane refers to HB-EGF in which secreted HB-EGF is bound to the cell membrane surface due to its heparin binding activity and electrostatic binding activity.

In the cell membrane, the substance to which secretory HB-EGF binds may be any substance that exists on the cell membrane and to which secretory HB-EGF binds, and is specifically a polysaccharide, more preferably Examples thereof include glycosaminoglycans, and particularly preferable examples include heparan sulfate.
HB-EGF has an activity of binding to diphtheria toxin, EGF receptor ErbB1 or ErbB4.
Examples of membrane-type HB-EGF include the following proteins (a), (b), and (c).
(a) a protein comprising the amino acid sequence represented by SEQ ID NO: 2;
(b) a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added in the amino acid sequence represented by SEQ ID NO: 2 and having an activity of binding diphtheria toxin;
(c) a protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 2 and to which diphtheria toxin binds;
Examples of the secreted HB-EGF include the following proteins (a), (b), and (c).
(a) a protein consisting of the amino acid sequence represented by SEQ ID NO: 3, 4 or 5;
(b) in the amino acid sequence represented by SEQ ID NO: 3, 4 or 5, consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added, and the EGF receptor ErbB1 or ErbB4 A protein having an activity of binding to;
(c) a protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 3, 4 or 5, and binding to the EGF receptor ErbB1 or ErbB4;
It consists of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added in the amino acid sequence represented by SEQ ID NO: 2, 3, 4 or 5, and is a diphtheria toxin or EGF receptor ErbB1 or ErbB4 Proteins that bind to are molecular cloning 2nd edition, Current Protocols in Molecular Biology, Nucleic Acids Research, 10, 6487 (1982), Proceedings National Academic・ Science USA (Proc. Natl. Acad. Sci.) USA, 79, 6409 (1982), Gene, 34, 315 (1985), etc. Tan which can be obtained by introducing a site-specific mutation into DNA encoding a protein having the amino acid sequence represented by SEQ ID NO: 2, 3, 4 or 5. It means click quality. The number of amino acids to be deleted, substituted, inserted and / or added is one or more, and the number is not particularly limited. For example, it is 1 to several tens, preferably 1 to 20, more preferably 1 to 10, and further preferably 1 to 5.

  Further, the protein having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 2, 3, 4 or 5 and binding to diphtheria toxin, EGF receptor ErbB1 or ErbB4 is SEQ ID NO: 2, At least 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, particularly preferably 97% or more, and most preferably 99% or more with a protein having the amino acid sequence described in 3, 4, or 5 It is a protein having an activity of binding to diphtheria toxin, EGF receptor ErbB1 or ErbB4 and having a homology of at least%.

  Unless otherwise specified, the homology value is a value calculated using a homology search program known to those skilled in the art, and the base sequence is BLAST [Journal of Molecular Biology (J. Mol. Biol.), 215, 403 (1990)], and the like are calculated using default parameters. For the amino acid sequence, BLAST2 [Nucleic Acid Res., 25, 3389 (1997)]; Genome Research, 7, 649 (1997); http: //www.ncbi There are numerical values calculated using default parameters in .nlm.nih.gov / Education / BLASTinfo / infomation3.html. Default parameters are 5 if G (Cost to open gap) is a base sequence, 11 if an amino acid sequence, 2 if -E (Cost to extend gap) is a base sequence, 1 if an amino acid sequence, -q (penalty for nucleotide mismatch) is -3, -r (reward for nucleotide match) is 1, -e (expect value) is 10, and -W (wordsize) is a base sequence, 11 residues, amino acid residues 3 residues, 20 if -y (Dropoff (X) for blast extemsions in bits) is blastn, 25 for programs other than blastn (http://www.ncbi.nlm.nih.gov/ blastcgihelp.html). Moreover, as an amino acid sequence analysis software, FASTA [Methods in Enzymology, 183, 63 (1990)] can be mentioned.

The antibody of the present invention is a monoclonal antibody that binds to HB-EGF, membrane-type HB-EGF, and secretory HB-EGF bound to the cell membrane, and includes HB-EGF, membrane-type HB- It includes monoclonal antibodies that bind to the epidermal growth factor-like domain (EGF-like domain) of EGF and secretory HB-EGF.
Specific examples of the EGF-like domain include a polypeptide having the amino acid sequence represented by SEQ ID NO: 4 or 5.

The monoclonal antibody that binds to the EGF-like domain includes a monoclonal antibody that inhibits the binding between secretory HB-EGF and HB-EGF receptor.
Examples of antibodies that inhibit the binding between secretory HB-EGF and HB-EGF receptor include monoclonal antibodies that bind to the binding region between secretory HB-EGF and HB-EGF receptor or diphtheria toxin.

The antibody of the present invention includes an antibody having neutralizing activity against secretory HB-EGF. In the present invention, the neutralizing activity refers to an activity that suppresses the biological activity of secreted HB-EGF, and includes, for example, an activity that suppresses cell proliferation of cells expressing the HB-EGF receptor.
Specific examples of the antibody of the present invention include a monoclonal antibody that binds to an epitope comprising at least one amino acid among the 115th to 147th amino acids of the polypeptide having the amino acid represented by SEQ ID NO: 2, preferably the 133rd Monoclonal antibody that binds to an epitope comprising at least one amino acid from 147 to 147, more preferably 115, 122, 124, 125, 127, 129, 133, 135, 141, and Monoclonal antibody that binds to an epitope comprising at least one amino acid among the 147th amino acids, more preferably, to an epitope comprising at least the 133th and 135th amino acids among the 133th, 135th, and 147th amino acids Monoclonal antibodies, most preferably amino acids at positions 133, 135 and 147 Such as monoclonal antibodies that bind to non-epitope, and the like.

More specifically, it binds to an epitope to which a monoclonal antibody produced by hybridoma KM3566 (FERM BP-10490), hybridoma KM3567 (FERM BP-10573), and a monoclonal antibody produced by hybridoma KM3579 (FERM BP-10491) binds. Monoclonal antibodies are exemplified as the antibodies of the present invention.
Specific examples of the antibody having neutralizing activity include a monoclonal antibody that binds to an epitope comprising amino acids 133, 135 and 147 of the polypeptide having the amino acid represented by SEQ ID NO: 2.

Examples of the monoclonal antibody of the present invention include antibodies produced by hybridomas and recombinant antibodies.
A hybridoma refers to a cell that produces a monoclonal antibody having a desired antigen specificity obtained by cell fusion of a B cell obtained by immunizing a mammal other than a human with an antigen and a myeloma cell.

The recombinant antibody includes antibodies produced by genetic recombination, such as human chimeric antibodies, humanized antibodies, human antibodies or antibody fragments thereof. A recombinant antibody having characteristics of a monoclonal antibody, low antigenicity, and extended blood half-life is preferable as a therapeutic agent.
Specific examples of the recombinant antibody of the present invention include the amino acid sequences in which CDR1, CDR2, and CDR3 of the VH of the antibody are represented by SEQ ID NOs: 12, 13, and 14, respectively, and CDR1, CDR2, and CDR3 of the antibody VL. Are recombinant antibodies comprising the amino acid sequences represented by SEQ ID NOs: 15, 16, and 17, respectively.

The gene recombinant antibody of the present invention includes antibodies produced by gene recombination, such as human chimeric antibodies, humanized antibodies, human antibodies or antibody fragments thereof.
A human chimeric antibody is an antibody comprising VH and VL of a non-human animal antibody and a heavy chain constant region (hereinafter referred to as CH) and a light chain constant region (hereinafter referred to as CL) of a human antibody. Say.

  The human chimeric antibody of the present invention can be prepared as follows. First, cDNA encoding VH and VL is obtained from a hybridoma producing HB-EGF, secretory HB-EGF, and monoclonal antibody that binds to membrane HB-EGF bound to the cell membrane. A human chimeric antibody expression vector is constructed by inserting the obtained cDNA into an expression vector for animal cells having genes encoding CH and CL of a human antibody, and introduced into an animal cell to express it. Type chimeric antibodies can be produced.

  The CH of the human chimeric antibody may be any as long as it belongs to human immunoglobulin (hereinafter referred to as hIg), but is preferably of the hIgG class, and more preferably hIgG1, hIgG2, hIgG3 belonging to the hIgG class, Any of the subclasses such as hIgG4 can be used. The CL of the human chimeric antibody may be any as long as it belongs to hIg, and those of κ class or λ class can be used.

  Specific examples of the human chimeric antibody of the present invention include a human chimeric antibody comprising the amino acid sequence represented by SEQ ID NO: 9 in the antibody VH and the amino acid sequence represented by SEQ ID NO: 11 in the antibody VL. can give. Furthermore, specific examples of the human chimeric antibody in which the antibody VH is represented by SEQ ID NO: 9 and the antibody VL is represented by SEQ ID NO: 10 include human chimeric antibody KM3966.

A humanized antibody refers to an antibody obtained by grafting the VH and VL CDR amino acid sequences of a non-human animal antibody to an appropriate position of the VH and VL of a human antibody. The CDR-grafted antibody and the reshaped-antibody Also called.
The humanized antibody of the present invention can be prepared as follows. First, the VH and VL CDRs of monoclonal antibodies from non-human animals that bind to HB-EGF, secretory HB-EGF and membrane HB-EGF bound to cell membranes produced by hybridomas A cDNA encoding a variable region transplanted to the VH and VL frameworks of human antibodies (hereinafter referred to as FR) is prepared. The prepared cDNA is inserted into an expression vector for animal cells having genes encoding human antibody CH and CL, respectively, to construct a humanized antibody expression vector. Next, the humanized antibody can be produced by introducing the prepared humanized antibody expression vector into animal cells to express the humanized antibody.

  Any amino acid sequences of FRs of VH and VL of human antibodies can be used as long as they are amino acid sequences of FRs of VH and VL derived from human antibodies. For example, human antibody VH and VL FR amino acid sequences registered in databases such as Protein Data Bank, or human antibodies described in Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991), etc. A common amino acid sequence of each subgroup of FR of VH and FR of VL is used.

The CH of the humanized antibody may be any as long as it belongs to hIg, but the hIgG class is suitable, and any of the subclasses such as hIgG1, hIgG2, hIgG3, and hIgG4 belonging to the hIgG class can be used. The CL of the humanized antibody may be any as long as it belongs to hIg, and those of κ class or λ class can be used.
As the humanized antibody of the present invention, specifically, the VH of the antibody is the amino acid sequence represented by SEQ ID NO: 22, or the 9th Ala in the amino acid sequence represented by SEQ ID NO: 22, the 20th Val, 30th Thr, 38th Arg, 41st Pro, 48th Met, 67th Arg, 68th Val, 70th Ile, 95th Tyr and 118th Val The amino acid sequence in which the amino acid residue is substituted with another amino acid residue and / or the VL of the antibody is the amino acid sequence represented by SEQ ID NO: 23, or the 15th Leu in the amino acid sequence represented by SEQ ID NO: 23, Humanized antibodies each containing an amino acid sequence in which at least one amino acid residue selected from 19th Ala, 21st Ile, 49th Pro and 84th Leu is substituted with another amino acid residue However, the number of modifications to be introduced is not particularly limited.

  For example, for the amino acid sequence of the antibody VH, the antibody VH is the 20th Val, 30th Thr, 38th Arg, 48th Met, 67th Arg in the amino acid sequence represented by SEQ ID NO: 22. 68th Val, 70th Ile, 95th Tyr, and 118th Val, preferably the 20th Val in the amino acid sequence in which the antibody VH is represented by SEQ ID NO: 22, 30th Thr, A humanized antibody having an amino acid sequence in which 48th Met, 68th Val, 70th Ile, 95th Tyr, and 118th Val are substituted with other amino acid residues.

Preferably 30th Thr, 48th Met, 68th Val, 70th Ile, 95th Tyr, preferably 30th Thr, 48th Met, 68th Val, 70th Ile A humanized antibody having an amino acid sequence substituted with another amino acid residue.
Preferably, a humanized antibody having an amino acid sequence in which the 30th Thr, 68th Val, 70th Ile, and 95th Tyr are substituted with other amino acid residues.

Preferably, a humanized antibody having an amino acid sequence in which the 30th Thr, 68th Val, and 70th Ile are substituted with other amino acid residues.
Preferred examples include humanized antibodies comprising an amino acid sequence in which the 30th Thr and the 70th Ile are substituted with other amino acid residues.
As the amino acid sequence of the antibody VH obtained as a result of the amino acid modification shown above, the 9th Ala in the amino acid sequence represented by SEQ ID NO: 22 is Thr, the 20th Val is Leu, and the 30th Thr To Arg, 38th Arg to Lys, 41st Pro to Thr, 48th Met to Ile, 67th Arg to Lys, 68th Val to Ala, 70th Ile to Leu An amino acid sequence in which at least one modification selected from modifications in which the 95th Tyr is replaced with Phe and the 118th Val is replaced with Leu is introduced.

  Specifically, as the amino acid sequence of VH into which 11 modifications have been introduced, the 9th Ala in the amino acid sequence represented by SEQ ID NO: 22 is Thr, the 20th Val is Leu, the 30th Thr to Arg, 38th Arg to Lys, 41st Pro to Thr, 48th Met to Ile, 67th Arg to Lys, 68th Val to Ala, 70th Ile Leu includes amino acid sequences in which the 95th Tyr is replaced with Phe and the 118th Val is replaced with Leu.

  Specifically, as the amino acid sequence of VH into which 10 modifications were introduced, the 9th Ala in the amino acid sequence represented by SEQ ID NO: 22 is Thr, the 20th Val is Leu, the 30th Thr to Arg, 38th Arg to Lys, 41st Pro to Thr, 48th Met to Ile, 67th Arg to Lys, 68th Val to Ala, 70th Ile Examples include amino acids obtained by substituting Leu and 95th Tyr with Phe.

  Specifically, as the amino acid sequence of VH into which nine modifications were introduced, the 9th Ala in the amino acid sequence represented by SEQ ID NO: 22 is Thr, the 20th Val is Leu, the 30th Thr to Arg, 41st Pro to Thr, 48th Met to Ile, 67th Arg to Lys, 68th Val to Ala, 70th Ile to Leu, and 95th Tyr The amino acid sequence in which is replaced with Phe, and the 20th Val to Leu, the 30th Thr to Arg, the 38th Arg to Lys, the 48th Met to Ile, the 67th Arg to Lys, 68 Examples include amino acid sequences in which the 1st Val is replaced with Ala, the 70th Ile is replaced with Leu, the 95th Tyr is replaced with Phe, and the 118th Val is replaced with Leu.

As an amino acid sequence of VH into which 8 modifications have been introduced, specifically, in the amino acid sequence represented by SEQ ID NO: 22
9th Ala to Thr, 20th Val to Leu, 30th Thr to Arg, 41st Pro to Thr, 48th Met to Ile, 68th Val to Ala, 70th An amino acid sequence obtained by substituting Ile for Leu and 95th Tyr for Phe,
20th Val to Leu, 30th Thr to Arg, 48th Met to Ile, 67th Arg to Lys, 68th Val to Ala, 70th Ile to Leu, 95th An amino acid sequence in which Tyr is replaced with Phe, and Val at position 118 is replaced with Leu, and
20th Val to Leu, 30th Thr to Arg, 38th Arg to Lys, 48th Met to Ile, 68th Val to Ala, 70th Ile to Leu, 95th Amino acid sequences in which Tyr is replaced with Phe and 118th Val is replaced with Leu, and the like.

As an amino acid sequence of VH into which seven modifications have been introduced, specifically, in the amino acid sequence represented by SEQ ID NO: 22
9th Ala to Thr, 30th Thr to Arg, 41st Pro to Thr, 48th Met to Ile, 68th Val to Ala, 70th Ile to Leu, and 95 An amino acid sequence in which the Tyr is replaced with Phe, and
20th Val to Leu, 30th Thr to Arg, 48th Met to Ile, 68th Val to Ala, 70th Ile to Leu, 95th Tyr to Phe, and 118 An amino acid sequence in which the second Val is replaced with Leu,
Etc.

As an amino acid sequence of VH into which six modifications have been introduced, specifically, in the amino acid sequence represented by SEQ ID NO: 22
9th Ala replaced with Thr, 30th Thr replaced with Arg, 48th Met replaced with Ile, 68th Val replaced with Ala, 70th Ile replaced with Leu, and 95th Tyr replaced with Phe. Amino acid sequence, and
20th Val replaced with Leu, 30th Thr replaced with Arg, 48th Met replaced with Ile, 68th Val replaced with Ala, 70th Ile replaced with Leu, and 95th Tyr replaced with Phe. Amino acid sequence,
Etc.

As an amino acid sequence of VH into which five modifications have been introduced, specifically, in the amino acid sequence represented by SEQ ID NO: 22
An amino acid sequence in which the 9th Ala is replaced by Thr, the 30th Thr is replaced by Arg, the 48th Met is replaced by Ile, the 68th Val is replaced by Ala, and the 70th Ile is replaced by Leu, and
Examples include an amino acid sequence in which the 30th Thr is replaced with Arg, the 48th Met is replaced with Ile, the 68th Val is replaced with Ala, the 70th Ile is replaced with Leu, and the 95th Tyr is replaced with Phe.

As an amino acid sequence of VH into which four modifications have been introduced, specifically, in the amino acid sequence represented by SEQ ID NO: 22
Amino acid sequence in which the 9th Ala is replaced with Thr, the 30th Thr with Arg, the 68th Val with Ala, and the 70th Ile with Leu,
An amino acid sequence in which the 30th Thr is replaced with Arg, the 48th Met with Ile, the 68th Val with Ala, and the 70th Ile with Leu,
An amino acid sequence in which the 20th Val is replaced with Leu, the 30th Thr with Arg, the 68th Val with Ala, and the 70th Ile with Leu,
An amino acid sequence in which the 30th Thr is replaced with Arg, the 38th Arg is replaced with Lys, the 68th Val is replaced with Ala, and the 70th Ile is replaced with Leu,
An amino acid sequence in which the 30th Thr is replaced with Arg, the 41st Pro with Thr, the 68th Val with Ala, and the 70th Ile with Leu,
An amino acid sequence in which the 30th Thr is substituted with Arg, the 67th Arg with Lys, the 68th Val with Ala, and the 70th Ile with Leu,
An amino acid sequence in which the 30th Thr is replaced with Arg, the 68th Val is replaced with Ala, the 70th Ile is replaced with Leu, and the 95th Tyr is replaced with Phe, and
An amino acid sequence in which the 30th Thr is replaced with Arg, the 68th Val is replaced with Ala, the 70th Ile is replaced with Leu, and the 118th Val is replaced with Leu,
Etc.

As an amino acid sequence of VH into which three modifications have been introduced, specifically, in the amino acid sequence represented by SEQ ID NO: 22
An amino acid sequence in which the 30th Thr is replaced with Arg, the 68th Val is replaced with Ala, and the 70th Ile is replaced with Leu,
An amino acid sequence in which the 9th Ala is replaced with Thr, the 30th Thr with Arg, and the 70th Ile with Leu,
An amino acid sequence in which the 20th Val is replaced with Leu, the 30th Thr with Arg, and the 70th Ile with Leu,
An amino acid sequence in which the 30th Thr is substituted with Arg, the 38th Arg with Lys, and the 70th Ile with Leu,
An amino acid sequence in which the 30th Thr is replaced with Arg, the 41st Pro with Thr, and the 70th Ile with Leu,
An amino acid sequence in which the 30th Thr is replaced with Arg, the 48th Met is replaced with Ile, and the 70th Ile is replaced with Leu,
An amino acid sequence in which the 30th Thr is substituted with Arg, the 67th Arg with Lys, and the 70th Ile with Leu,
An amino acid sequence in which the 30th Thr is replaced with Arg, the 70th Ile with Leu, and the 95th Tyr with Phe, and
An amino acid sequence in which the 30th Thr is replaced with Arg, the 70th Ile with Leu, and the 118th Val with Leu,
Etc.

As an amino acid sequence of VH into which two modifications are introduced, specifically, in the amino acid sequence represented by SEQ ID NO: 22
An amino acid sequence in which the 30th Thr is replaced with Arg and the 70th Ile is replaced with Leu,
Amino acid sequence in which the 9th Ala is replaced with Thr and the 70th Ile is replaced with Leu,
An amino acid sequence in which the 20th Val is replaced with Leu and the 70th Ile is replaced with Leu,
Amino acid sequence in which 38th Arg is replaced with Lys and 70th Ile is replaced with Leu,
Amino acid sequence in which the 41st Pro is replaced with Thr and the 70th Ile is replaced with Leu,
Amino acid sequence in which the 48th Met is replaced with Ile and the 70th Ile is replaced with Leu,
Amino acid sequence in which 67th Arg is replaced with Lys and 70th Ile is replaced with Leu,
Amino acid sequence in which 68th Val is replaced with Ala and 70th Ile is replaced with Leu,
An amino acid sequence in which the 70th Ile is replaced with Leu and the 95th Tyr with Phe;
Amino acid sequence in which 70th Ile is replaced with Leu and 118th Val is replaced with Leu,
Amino acid sequence in which the 9th Ala is replaced with Thr and the 30th Thr is replaced with Arg,
An amino acid sequence in which the 20th Val is replaced with Leu and the 30th Thr with Arg,
An amino acid sequence in which the 30th Thr is replaced with Arg, and the 38th Arg is replaced with Lys,
An amino acid sequence in which the 30th Thr is replaced with Arg and the 41st Pro is replaced with Thr,
An amino acid sequence in which the 30th Thr is replaced with Arg and the 48th Met is replaced with Ile,
An amino acid sequence in which the 30th Thr is replaced with Arg, and the 67th Arg is replaced with Lys,
An amino acid sequence in which the 30th Thr is replaced with Arg and the 68th Val is replaced with Ala,
An amino acid sequence in which the 30th Thr is replaced with Arg, and the 95th Tyr is replaced with Phe; and
An amino acid sequence in which the 30th Thr is replaced with Arg and the 118th Val is replaced with Leu,
Etc.

As an amino acid sequence of VH into which one modification has been introduced, specifically, in the amino acid sequence represented by SEQ ID NO: 22
Amino acid sequence with 9th Ala substituted with Thr, 20th Val substituted with Leu, 30th Thr substituted with Arg, 38th Arg substituted with Lys, 41st amino acid sequence Amino acid sequence in which Pro is replaced with Thr, amino acid sequence in which 48th Met is replaced with Ile, amino acid sequence in which 67th Arg is replaced with Lys, amino acid sequence in which 68th Val is replaced with Ala, 70th Ile Amino acid sequence in which is replaced with Leu, amino acid sequence in which 95th Tyr is replaced with Phe, and amino acid sequence in which 118th Val is replaced with Leu.

Examples of the VL of the antibody include amino acid sequences in which the 15th Leu, 19th Ala, 21st Ile, and 84th Leu in the amino acid sequence represented by SEQ ID NO: 23 are substituted.
Preferred examples include amino acid sequences in which the 19th Ala, 21st Ile, and 84th Leu are substituted.
As the amino acid sequence of VL obtained as a result of the above amino acid modification, the 15th Leu in the amino acid sequence represented by SEQ ID NO: 23 is Val, the 19th Ala is Val, the 21st Ile is Met, Examples include amino acid sequences into which at least one modification selected from a modification in which the 49th Pro is replaced with Ser and the 84th Leu is replaced with Val is introduced.

Specifically, as the amino acid sequence of VL introduced with five modifications, the 15th Leu in the amino acid sequence represented by SEQ ID NO: 23 is Val, the 19th Ala is Val, and the 21st Examples include amino acid sequences in which Ile is replaced by Met, 49th Pro is replaced by Ser, and 84th Leu is replaced by Val.
As an amino acid sequence of VL into which four modifications have been introduced, specifically, in the amino acid sequence represented by SEQ ID NO: 23
An amino acid sequence in which the 15th Leu is replaced with Val, the 19th Ala is replaced with Val, the 21st Ile is replaced with Met, and the 49th Pro is replaced with Ser.
An amino acid sequence in which the 15th Leu is replaced with Val, the 19th Ala is replaced with Val, the 21st Ile is replaced with Met, and the 84th Leu is replaced with Val.
An amino acid sequence in which the 15th Leu is replaced with Val, the 19th Ala is replaced with Val, the 49th Pro is replaced with Ser, and the 84th Leu is replaced with Val.
An amino acid sequence in which the 15th Leu is replaced by Val, the 21st Ile is replaced by Met, the 49th Pro is replaced by Ser, and the 84th Leu is replaced by Val, and
Amino acid sequence in which the 19th Ala is replaced with Val, the 21st Ile is replaced with Met, the 49th Pro is replaced with Ser, and the 84th Leu is replaced with Val.
Etc.

As an amino acid sequence of VL into which three modifications have been introduced, specifically, in the amino acid sequence represented by SEQ ID NO: 23
Amino acid sequence in which the 15th Leu is replaced with Val, the 19th Ala with Val, and the 21st Ile with Met,
Amino acid sequence in which the 15th Leu is replaced with Val, the 19th Ala is replaced with Val, and the 49th Pro is replaced with Ser.
An amino acid sequence in which the 15th Leu is replaced with Val, the 19th Ala is replaced with Val, and the 84th Leu is replaced with Val,
Amino acid sequence in which the 15th Leu is replaced with Val, the 21st Ile is replaced with Met, and the 49th Pro is replaced with Ser.
Amino acid sequence in which the 15th Leu is replaced with Val, the 21st Ile is replaced with Met, and the 84th Leu is replaced with Val.
An amino acid sequence in which the 15th Leu is replaced with Val, the 49th Pro is replaced with Ser, and the 84th Leu is replaced with Val.
Amino acid sequence in which the 19th Ala is replaced with Val, the 21st Ile is replaced with Met, and the 49th Pro is replaced with Ser.
Amino acid sequence in which the 19th Ala is replaced with Val, the 21st Ile is replaced with Met, and the 84th Leu is replaced with Val,
Amino acid sequence in which 19th Ala is replaced with Val, 49th Pro is replaced with Ser, and 84th Leu is replaced with Val, and
Amino acid sequence in which the 21st Ile is replaced with Met, the 49th Pro with Ser, and the 84th Leu with Val
Etc.

Specifically, the amino acid sequence of VL introduced with two modifications is the amino acid sequence represented by SEQ ID NO: 23.
An amino acid sequence in which the 15th Leu is replaced with Val and the 19th Ala is replaced with Val,
An amino acid sequence in which the 15th Leu is replaced with Val and the 21st Ile is replaced with Met,
Amino acid sequence in which the 15th Leu is replaced with Val and the 49th Pro is replaced with Ser,
An amino acid sequence in which the 15th Leu is replaced with Val and the 84th Leu is replaced with Val,
Amino acid sequence in which the 19th Ala is replaced with Val and the 21st Ile is replaced with Met,
Amino acid sequence in which 19th Ala is replaced with Val and 49th Pro is replaced with Ser,
Amino acid sequence in which 19th Ala is replaced with Val and 84th Leu is replaced with Val,
Amino acid sequence in which 21st Ile is replaced with Met and 49th Pro is replaced with Ser,
Amino acid sequence in which 21st Ile is replaced with Met and 84th Leu is replaced with Val,
And amino acid sequence in which the 49th Pro is replaced with Ser, and the 84th Leu is replaced with Val,
Etc.

As an amino acid sequence of VL into which one modification has been introduced, specifically, in the amino acid sequence represented by SEQ ID NO: 23
An amino acid sequence in which the 15th Leu is replaced with Val, an amino acid sequence in which the 19th Ala is replaced with Val, an amino acid sequence in which the 21st Ile is replaced with Met, an amino acid sequence in which the 49th Pro is replaced with Ser, and 84 An amino acid sequence in which the second Leu is replaced with Val, and the like.

Specific examples of the humanized antibody of the present invention include humanized antibodies whose variable regions have the amino acid sequences represented by SEQ ID NOs: 22 and 23.
A human antibody originally refers to an antibody naturally present in the human body, but a human antibody phage library and human antibody production produced by recent advances in genetic engineering, cell engineering, and developmental engineering techniques Also included are antibodies obtained from transgenic animals. The antibody present in the human body can be isolated and cultured, for example, by isolating human peripheral blood lymphocytes, infecting and immortalizing EB virus, etc., and cloning the antibody-producing lymphocytes. The antibody can be purified from the supernatant. The human antibody phage library is a library in which antibody fragments such as Fab and scFv are expressed on the phage surface by inserting an antibody gene prepared from human B cells into the phage gene. From the library, phages expressing an antibody fragment having a desired antigen-binding activity on the surface can be collected using the binding activity to the substrate on which the antigen is immobilized as an index. The antibody fragment can be further converted into a human antibody molecule comprising two complete heavy chains and two complete light chains by genetic engineering techniques. A human antibody-producing transgenic animal means an animal in which a human antibody gene is integrated into the genome gene of a host animal. Specifically, for example, a human antibody-producing transgenic mouse can be produced by introducing a human antibody gene into a mouse ES cell, transplanting the ES cell into an early mouse embryo, and generating the mouse embryo. A human antibody production method from a human antibody-producing transgenic animal is obtained by obtaining and culturing a human antibody-producing hybridoma by a hybridoma production method performed in a normal non-human animal. Can be generated and accumulated.

An antibody or antibody fragment thereof in which one or more amino acids are deleted, added, substituted or inserted in the amino acid sequence constituting the antibody or antibody fragment described above and has the same activity as the antibody or antibody fragment thereof described above, It is included in the antibody of the present invention or an antibody fragment thereof.
The number of amino acids to be deleted, substituted, inserted and / or added is one or more, and the number is not particularly limited. Molecular Cloning 2nd Edition, Current Protocols in Molecular Biology, Nucleic Acids Research, 10, 6487 (1982), Proc. Natl. Acad. Sci., USA, 79, 6409 (1982), Gene, 34, 315 (1985), Nucleic Acids Research, 13, 4431 (1985), Proc. Natl Acad. Sci USA, 82, 488 (1985) is a number that can be deleted, substituted, or added by a well-known technique such as site-directed mutagenesis described in, for example. For example, it is 1 to several tens, preferably 1 to 20, more preferably 1 to 10, and further preferably 1 to 5.

  Deletion, substitution, insertion or addition of one or more amino acid residues in the amino acid sequence of the above antibody indicates the following. It means that there is a deletion, substitution, insertion or addition of one or more amino acid residues in any and one or more amino acid sequences in the same sequence. In addition, deletion, substitution, insertion or addition may occur simultaneously, and the amino acid residue to be substituted, inserted or added may be either a natural type or a non-natural type. Natural amino acid residues include L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine , L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine and L-cysteine.

The preferred examples of the amino acid residues that can be substituted with each other are shown below. Amino acid residues contained in the same group can be substituted for each other.
Group A: leucine, isoleucine, norleucine, valine, norvaline, alanine, 2-aminobutanoic acid, methionine, O-methylserine, t-butylglycine, t-butylalanine, cyclohexylalanine Group B: aspartic acid, glutamic acid, isoaspartic acid, Isoglutamic acid, 2-aminoadipic acid, 2-aminosuberic acid Group C: asparagine, glutamine Group D: lysine, arginine, ornithine, 2,4-diaminobutanoic acid, 2,3-diaminopropionic acid Group E: proline, 3 -Hydroxyproline, 4-hydroxyproline Group F: serine, threonine, homoserine Group G: phenylalanine, tyrosine Examples of the antibody fragment of the present invention include Fab, Fab ', F (ab') ₂ , scFv, diabody, dsFv and the like. It is done.

Fab is obtained by treating an IgG antibody molecule with the proteolytic enzyme papain. Among these fragments (cleaved at the 224th amino acid residue of the H chain), an antibody fragment having an antigen-binding activity of about 50,000 molecular weight, in which about half of the N-terminal side of the H chain and the entire L chain are bound by a disulfide bond It is.
The Fab of the present invention can be obtained by treating an antibody with the proteolytic enzyme papain. Alternatively, the Fab is expressed and produced by inserting the DNA encoding the Fab of the antibody into a prokaryotic expression vector or a eukaryotic expression vector, and introducing the vector into a prokaryotic or eukaryotic organism. Can do.

F (ab ') ₂ is a fragment obtained by treating an IgG antibody molecule with the protease enzyme pepsin (cleaved at the 234th amino acid residue of the H chain), and Fab represents a disulfide bond in the hinge region. It is an antibody fragment having an antigen-binding activity with a molecular weight of about 100,000, which is slightly larger than those bound via the intercalation.
The F (ab ′) ₂ of the present invention can be obtained by treating an antibody with the proteolytic enzyme pepsin. Alternatively, Fab ′ described below can be prepared by thioether bond or disulfide bond.

Fab ′ is an antibody fragment having a molecular weight of about 50,000 and having an antigen binding activity obtained by cleaving the disulfide bond in the hinge region of F (ab ′) ₂ .
The Fab ′ of the present invention can be obtained by treating F (ab ′) ₂ with a reducing agent dithiothreitol. Alternatively, the DNA encoding the Fab ′ fragment of the antibody is inserted into a prokaryotic expression vector or eukaryotic expression vector, and Fab ′ is expressed by introducing the vector into a prokaryotic or eukaryotic organism. can do.

  scFv is a VH-P-VL or VL-P-VH polypeptide in which one VH and one VL are linked using an appropriate peptide linker (hereinafter referred to as P) and has antigen-binding activity. It is an antibody fragment having The scFv of the present invention obtains cDNA encoding the antibody VH and VL, constructs the DNA encoding scFv, inserts the DNA into a prokaryotic expression vector or eukaryotic expression vector, and the expression vector ScFv can be expressed and produced by introducing into a prokaryote or eukaryote.

  A diabody is an antibody fragment obtained by dimerizing scFv and is an antibody fragment having a bivalent antigen-binding activity. The bivalent antigen binding activity can be the same, or one can be a different antigen binding activity. The diabody of the present invention obtains cDNA encoding the antibody VH and VL, constructs the DNA encoding scFv so that the length of the amino acid sequence of the linker is 8 residues or less, and uses the DNA for prokaryotes A diabody can be expressed and produced by inserting it into an expression vector or an eukaryotic expression vector and introducing the expression vector into a prokaryotic or eukaryotic organism.

dsFv refers to a polypeptide in which one amino acid residue in each of VH and VL is substituted with a cysteine residue, which are linked via a disulfide bond between the cysteine residues. The amino acid residue substituted for the cysteine residue can be selected based on the three-dimensional structure prediction of the antibody according to the method shown by Reiter et al. (Protein Engineering, 7 , 697-704, 1994). The dsFv of the present invention obtains cDNA encoding the antibody VH and VL, constructs a DNA encoding dsFv, inserts the DNA into a prokaryotic expression vector or eukaryotic expression vector, and the expression vector Is introduced into prokaryotes or eukaryotes, and dsFv can be expressed and produced.

Derivatives of antibodies in which a radioisotope, protein or drug is bound to the above-described antibody or antibody fragment are also encompassed in the present invention.
The derivative of the antibody of the present invention is the N-terminal of the H chain or L chain of the antibody or the antibody fragment that specifically binds to HB-EGF, membrane-type HB-EGF and secretory HB-EGF bound to the cell membrane. The drug is bound to the side or C-terminal side, appropriate substituents or side chains in the antibody, and sugar chains in the antibody by chemical techniques (Introduction to Antibody Engineering, Osamu Kanmitsu, Jinshoshokan, 1994) Can be manufactured.

Alternatively, it encodes an antibody that specifically binds to HB-EGF, membrane-type HB-EGF and secretory HB-EGF bound to the cell membrane or DNA encoding the antibody fragment and a drug such as a protein to be bound. It can also be produced by genetic engineering techniques in which DNA is ligated and inserted into an expression vector, and the expression vector is introduced into a host cell.
Examples of the drug include chemotherapeutic agents, antibody drugs, immunostimulants such as cytokines, radioisotopes, and immunoadjuvants.

Furthermore, the agent to be bound to the antibody or the antibody fragment may be in the form of a prodrug. The prodrug in the present invention refers to a drug that is chemically modified by an enzyme or the like present in a tumor environment and converted into a substance having an action of damaging cancer cells.
As chemotherapeutic agents, alkylating agents, nitrosourea agents, antimetabolites, anticancer antibiotics, plant-derived alkaloids, topoisomerase inhibitors, hormone therapy agents, hormone antagonists, aromatase inhibitors, P glycoprotein inhibitors, Any chemotherapeutic agent such as platinum complex derivatives, M phase inhibitors, kinase inhibitors, etc. is included. Chemotherapeutic agents include amifostine (ethiol), cisplatin, dacarbazine (DTIC), dactinomycin, mechlorethamine (nitrogen mustard), streptozocin, cyclophosphamide, ifosfamide, carmustine (BCNU), lomustine (CCNU), doxorubicin (Adriamycin), doxorubicin lipo (doxyl), epirubicin, gemcitabine (gemzar), daunorubicin, daunorubicin lipo (daunosome), procarbazine, mitomycin, cytarabine, etoposide, methotrexate, 5-fluorouracil, fluorouracil, vinblastine, vinblastine, vinblastine , Estramustine, paclitaxel (taxol), docetaxel (taxote) ), Aldesleukin, asparaginase, busulfan, carboplatin, oxaliplatin, nedaplatin, cladribine, camptothecin, CPT-11, 10-hydroxy-7-ethyl-camptothecin (SN38), floxuridine, fludarabine, hydroxyurea, ifosfamide, idarubicin, Mesna, irinotecan, nogitane, mitoxantrone, topotecan, leuprolide, megestrol, melphalan, mercaptopurine, hydroxycarbamide, pricamycin, mitotan, pegaparagase, pentostatin, pipobroman, streptozocin, tamoxifen, goserelin, leuprorenin , Flutamide, teniposide, test lactone, thioguanine, thiotepa, uracil mustard, vinorelbine, chlorambucil, high Locotisone, prednisolone, methylprednisolone, vindesine, nimustine, semstin, capecitabine, tomdex, azacitidine, UFT, oxaloplatine, gefitinib (Iressa), imatinib (STI571), erlotinib, Flt3 inhibitor, receptorvascularVEGFR Agents, fibroblast growth factor (FGFR) inhibitors, EGFR inhibitors (Iressa, Tarceva, etc.) Radicicol, 17-allylamino-17-demethoxygeldanamycin, rapamycin, amsacrine, all-trans retinoic acid, thalidomide, anastrozole , Fadrozole, letrozole, exemestane, gold thiomalate, D-penicillamine, bucillamine, azathioprine, mizoribine, cyclosporine, rapamycin, hydrocortisone, bexarotene ( -Gretin), tamoxifen, dexamethasone, progestins, estrogens, anastrozole (arimidex), leuprin, aspirin, indomethacin, celecoxib, azathioprine, penicillamine, gold thiomalate, chlorpheniramine maleate, chloropheniramine, clemacytine, tretinoin, bexarotene , Arsenic, bortezomib, allopurinol, gemtuzumab, ibritumomab tiuxetan, 131 tositemab, targretin, ONTAK, ozogamine, clarithroma machine, leucovorin, ifasfamide, ketoconazole, aminoglutethimide, suramin and methotrexate.

The chemotherapeutic agent and the antibody can be combined with each other by binding between the chemotherapeutic agent and the amino group of the antibody via glutaraldehyde, the amino group of the chemotherapeutic agent and the carboxyl group of the antibody via water-soluble carbodiimide. And the like.
Antibody drugs include antibodies against antigens whose apoptosis is induced by antibody binding, or antibodies against antigens involved in the pathogenesis of tumors such as tumor cell growth and metastasis, as well as antibodies that regulate immune function, lesion sites Examples thereof include antibodies that inhibit angiogenesis.

Antigens that induce apoptosis by antibody binding include Cluster of differentiation (CD) 19, CD20, CD21, CD22, CD23, CD24, CD37, CD53, CD72, CD73, CD74, CDw75, CDw76, CD77, CDw78 CD79a, CD79b, CD80 (B7.1), CD81, CD82, CD83, CDw84, CD85, CD86 (B7.2), human leukocyte antigen (HLA) -Class II, EGFR and the like.
Antibody antigens that regulate immune function include CD4, CD40, CD40 ligand, B7 family molecules (CD80, CD86, CD274, B7-DC, B7-H2, B7-H3, B7-H4), B7 family molecule ligands (CD28, CTLA-4, ICOS, PD-1, BTLA), OX-40, OX-40 ligand, CD137, tumor necrosis factor (TNF) receptor family molecules (DR4, DR5, TNFR1, TNFR2), TNF-related apoptosis-inducing ligand receptor (TRAIL) family molecule, receptor family of TRAIL family molecules (TRAIL-R1, TRAIL-R2, TRAIL-R3, TRAIL-R4), receptor activator of nuclear factor kappa B ligand (RANK), RANK ligand , CD25, folate receptor 4, cytokine [interleukin-1α (hereinafter referred to as interleukin), IL-1β, IL-4, IL-5, IL-6, IL-10, IL-13, transforming growth factor ( TGF) β, TNFα, etc.], receptors for these cytokines, chemokines (SLC, ELC, I-309, TARC, MDC, CTACK, etc.), and receptors for these chemokines are preferred.

Antibody antigens that inhibit angiogenesis at the lesion include vascular endothelial growth factor (VEGF), Angiopoietin, fibroblast growth factor (FGF), EGF, platelet-derived growth factor (PDGF), and insulin-like growth factor (IGF). Erythropoietin (EPO), TGFβ, IL-8, Ephilin, SDF-1, etc., and their receptors.
The immunostimulant may be any cytokine that enhances cells such as NK cells, macrophages, and neutrophils. Specific examples include interferon (hereinafter referred to as INF) -α, INF-β, INF-γ, IL-2, IL-12, IL-15, IL-18, IL-21, IL-23, granulocyte stimulating factor (G-CSF), granulocyte / macrophage stimulating factor (GM-CSF), Examples include macrophage stimulating factor (M-CSF). In addition, natural products known as immunostimulants may be used, and specific examples include β (1 → 3) glucan (lentinan, schizophyllan), α galactosylceramide (KRN7000), bacterial powder (Picibanil, BCG), and bacterial cell extract (krestin). Examples of radioactive isotopes include ¹³¹ I, ¹²⁵ I, ⁹⁰ Y, ⁶⁴ Cu, ¹⁹⁹ Tc, ⁷⁷ Lu, and ²¹¹ At. The radioisotope can be directly bound to the antibody by the chloramine T method or the like. Further, a substance that chelates a radioisotope may be bound to the antibody. Examples of chelating agents include methylbenzyldiethylene-triaminepentaacetic acid (MX-DTPA).

In the present invention, the antibody of the present invention can be administered in combination with one or more other drugs, or irradiation can be combined. Other drugs include the above-mentioned chemotherapeutic agents, antibody drugs, immunostimulants such as cytokines, and the like.
Examples of antibody drugs include antibodies against antigens that can be the above targets, such as EGFR antibodies (Cetuximab, Panitumumab, Matuzumab, etc.) and the like.

Radiation irradiation includes photon (electromagnetic wave) irradiation such as X-ray and γ-ray, particle beam irradiation such as electron beam, proton beam and heavy particle beam.
The method of administration in combination may be simultaneous administration with the antibody of the present invention, or may be administered before or after administration of the antibody of the present invention.
The present invention is described in detail below.

1. Production method of recombinant antibody (1) Preparation of antigen Including cDNA encoding secretory HB-EGF or partial length of secreted HB-EGF (hereinafter sometimes referred to as secreted HB-EGF) A secretory HB-EGF or a secretory HB-EGF partial fragment can be obtained by introducing and expressing an expression vector in Escherichia coli, yeast, insect cells, animal cells or the like. Moreover, HB-EGF in the extracellular region can be purified from cells expressing HB-EGF by protease treatment. Secreted HB-EGF can also be purified from cultured human tumor cells, human tissues, etc. that express a large amount of secreted HB-EGF. Furthermore, a synthetic peptide having a secretory HB-EGF partial sequence can be prepared and used as an antigen.

  Specifically, the secretory HB-EGF used in the present invention includes Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989) and Current Protocols in Molecular Biology, John Wiley & Sons (1987- 1997) and the like, for example, by the following method, DNA encoding the same can be expressed in a host cell and produced.

  First, a recombinant vector is prepared by inserting a full-length cDNA downstream of a promoter of an appropriate expression vector. In this case, if necessary, a DNA fragment having an appropriate length containing a portion encoding HB-EGF based on the full-length cDNA may be prepared, and the DNA fragment may be used in place of the full-length cDNA. . Next, a transformant producing HB-EGF can be obtained by introducing the recombinant vector into a host cell suitable for the expression vector.

Any host cell can be used as long as it can express the target gene, such as Escherichia coli and animal cells.
As the expression vector, one that can autonomously replicate in the host cell to be used or can be integrated into the chromosome and contains an appropriate promoter at a position where DNA encoding secretory HB-EGF can be transcribed is used. .

  When a prokaryote such as E. coli is used as a host cell, the recombinant vector containing the DNA encoding HB-EGF used in the present invention is capable of autonomous replication in prokaryote, and at the same time a promoter, A vector containing a ribosome binding sequence, DNA used in the present invention, and a transcription termination sequence is preferred. The recombinant vector may further contain a gene that controls the promoter.

  Examples of expression vectors include pBTrp2, pBTac1, pBTac2 (all manufactured by Roche Diagnostics), pKK233-2 (manufactured by Pharmacia), pSE280 (manufactured by Invitrogen), pGEMEX-1 (manufactured by Promega), pQE-8 ( QIAGEN), pKYP10 (Japanese Patent Laid-open No. 58-110600), pKYP200 [Agricultural Biological Chemistry, 48, 669 (1984)], pLSA1 [Agric. Biol. Chem., 53, 277 (1989)], pGEL1 [Proc. Natl. Acad. Sci. USA, 82, 4306 (1985)], pBluescript II SK (-) (Stratagene), pTrs30 [prepared from E. coli JM109 / pTrS30 (FERM BP-5407)], pTrs32 [E. coli JM109 / pTrS32 (Prepared from (FERM BP-5408)], pGHA2 [prepared from E. coli IGHA2 (FERM BP-400), JP 60-221091], pGKA2 [prepared from E. coli IGKA2 (FERM BP-6798), JP 60-221091 ], PTerm2 (US4686191, US4939094, US5160735), pSupex, pUB110, pTP5, pC194, pEG400 [J. Bacteriol., 172, 2392 (1990)], pGEX (Pharmacia), pET system (Novagen), pME18SFL3 Etc. It can gel.

  Any promoter can be used as long as it can function in the host cell to be used. For example, promoters derived from Escherichia coli or phages such as trp promoter (Ptrp), lac promoter, PL promoter, PR promoter, T7 promoter and the like can be mentioned. In addition, artificially designed and modified promoters such as a tandem promoter, tac promoter, lacT7 promoter, let I promoter, etc. in which two Ptrps are connected in series can also be used.

  Moreover, it is preferable to use a plasmid in which the distance between the Shine-Dalgarno sequence, which is a ribosome binding sequence, and the start codon is adjusted to an appropriate distance (for example, 6 to 18 bases) as the recombinant vector. In the base sequence of the DNA encoding secretory HB-EGF used in the present invention, the base can be substituted so as to be an optimal codon for expression in the host, and thus the target secreted HB-B -The production rate of EGF can be improved. Furthermore, a transcription termination sequence is not necessarily required for gene expression in the above recombinant vector, but it is preferable to place a transcription termination sequence immediately below the structural gene.

Host cells include microorganisms belonging to the genus Escherichia, such as E. coli XL1-Blue, E. coli XL2-Blue, E. coli DH1, E. coli DH5α, E. coli BL21 (DE3), E. coli MC1000, E. coli KY3276, E. coli W1485, E. coli JM109, E. coli HB101 E. coli No. 49, E. coli W3110, E. coli NY49 and the like.
As a method for introducing the recombinant vector, any method can be used as long as it is a method for introducing DNA into the host cell. For example, a method using calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110 ( 1972)], Gene, 17, 107 (1982) and Molecular & General Genetics, 168, 111 (1979).

  When animal cells are used as hosts, examples of expression vectors include pcDNAI, pcDM8 (commercially available from Funakoshi), pAGE107 [JP-A-3-22979; Cytotechnology, 3, 133, (1990)], pAS3-3 (special (Kaihei 2-227075), pCDM8 [Nature, 329, 840, (1987)], pcDNAI / Amp (Invitrogen), pREP4 (Invitrogen), pAGE103 [J. Biochemistry, 101, 1307 (1987)], pAGE210 , PME18SFL3 and the like.

  Any promoter can be used as long as it can function in animal cells. For example, cytomegalovirus (CMV) IE (immediate early) gene promoter, SV40 early promoter, retrovirus promoter , Metallothionein promoter, heat shock promoter, SRα promoter and the like. In addition, an IE gene enhancer of human CMV may be used together with a promoter.

Examples of host cells include human cells such as Namalwa cells, COS cells that are monkey cells, CHO cells that are Chinese hamster cells, and HBT5637 (Japanese Patent Laid-Open No. 63-299).
As a method for introducing a recombinant vector, any method can be used as long as it is a method for introducing DNA into animal cells. For example, electroporation method [Cytotechnology, 3, 133 (1990)], calcium phosphate method (Japanese Patent Laid-open No. Hei 2). -227075), lipofection method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)] and the like.

  As a gene expression method, in addition to direct expression, secretory production, fusion protein expression, etc. are performed according to the method described in Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989), etc. be able to. When expressed in cells derived from eukaryotes, secretory HB-EGF to which sugars or sugar chains have been added can be obtained.

A secretory HB-EGF can be produced by culturing the transformant obtained as described above in a medium, producing and accumulating the HB-EGF in the culture, and collecting it from the culture. The method of culturing the transformant in a medium can be performed according to a usual method used for culturing a host.
When culturing a microorganism transformed with a recombinant vector using an inducible promoter as a promoter, an inducer may be added to the medium as necessary. For example, when cultivating a microorganism transformed with a recombinant vector using the lac promoter, when culturing a microorganism transformed with isopropyl-β-D-thiogalactopyranoside or the like using a recombinant vector using the trp promoter. Indoleacrylic acid or the like may be added to the medium.

As a medium for culturing a transformant obtained by using an animal cell as a host, a commonly used RPMI1640 medium [The Journal of the American Medical Association, 199, 519 (1967)], Eagle's MEM medium [Science, 122 , 501 (1952)], Dulbecco's modified MEM medium [Virology, 8, 396 (1959)], 199 medium [Proc. Soc. Exp. Biol. Med., 73, 1 (1950)] or fetal calf serum in these media Etc. can be used. The culture is usually carried out for 1 to 7 days under conditions such as pH 6 to 8, 30 to 40 ° C., and the presence of 5% CO ₂ . Moreover, you may add antibiotics, such as kanamycin and penicillin, to a culture medium as needed during culture | cultivation.

As described above, a transformant derived from a microorganism, animal cell, or the like that possesses a recombinant vector incorporating a DNA encoding secretory HB-EGF used in the present invention is cultured according to a normal culture method, and the HB- Secreted HB-EGF used in the present invention can be produced by producing and accumulating EGF and collecting it from the culture.
As a gene expression method, in addition to direct expression, secretory production, fusion protein expression, etc. are performed according to the method described in Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989), etc. be able to.

The secretory HB-EGF can be produced in a host cell, secreted outside the host cell, or produced on the host cell membrane. The host cell used and the secreted form produced. By changing the structure of HB-EGF, an appropriate method can be selected.
When secreted HB-EGF is produced in the host cell or on the host cell outer membrane, the method of Paulson et al. [J. Biol. Chem., 264, 17619 (1989)], the method of Rowe et al. [Proc. Natl. Acad. Sci., USA, 86, 8227 (1989), Genes Develop., 4, 1288 (1990)], or Japanese Patent Application Laid-Open No. 05-336963, WO94 / 23021, etc. Can be actively secreted out of the host cell.

  Further, in accordance with the method described in JP-A-2-27075, the production amount can be increased using a gene amplification system using a dihydrofolate reductase gene or the like. Secreted HB-EGF can be isolated and purified as follows, for example. If secreted HB-EGF is expressed in a dissolved state in the cells, the cells are collected by centrifugation after culturing, suspended in an aqueous buffer, and then subjected to an ultrasonic crusher, French press, Manton Gaurin homogenizer, and dynomill. The cells are disrupted by, for example, a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, an ordinary enzyme isolation and purification method, that is, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, diethylamino Anion exchange chromatography using a resin such as ethyl (DEAE) -Sepharose and DIAION HPA-75 (Mitsubishi Chemical), and cation exchange chromatography using a resin such as S-Sepharose FF (Pharmacia) Methods such as electrophoresis, hydrophobic chromatography using resin such as butyl sepharose, phenyl sepharose, gel filtration using molecular sieve, affinity chromatography, chromatofocusing, isoelectric focusing etc. A purified sample can be obtained by using alone or in combination.

  In addition, when secreted HB-EGF is expressed in the form of an insoluble substance in the cell, similarly, the cell is recovered and then disrupted and centrifuged to obtain the HB-EGF insoluble substance as a precipitate fraction. to recover. The recovered insoluble matter of the protein is solubilized with a protein denaturant. By diluting or dialyzing the solubilized solution, the protein is returned to a normal three-dimensional structure, and then a purified preparation of secreted HB-EGF can be obtained by the same isolation and purification method as described above.

When a derivative such as secreted HB-EGF or a sugar-modified product thereof is secreted extracellularly, the derivative such as HB-EGF or a sugar-modified product thereof can be recovered in the culture supernatant. That is, a soluble fraction is obtained by treating the culture by a technique such as centrifugation as described above, and a purified preparation is obtained from the soluble fraction by using the same isolation and purification method as described above. be able to.
The secretory HB-EGF used in the present invention can also be produced by chemical synthesis methods such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (t-butyloxycarbonyl method). Chemical synthesis can also be performed using peptide synthesizers such as Advanced ChemTech, Perkin Elmer, Pharmacia, Protein Technology Instrument, Synthecell-Vega, PerSeptive, Shimadzu Corporation.
(2) Immunization of animals and preparation of antibody-producing cells
Mice, rats, or hamsters 3 to 20 weeks old are immunized with the antigen prepared as described above, and antibody-producing cells in the spleen, lymph nodes, and peripheral blood of the animals are collected. There is also a method of using an HB-EGF knockout mouse as an immunized animal when the immunogenicity is low and a sufficient increase in antibody titer is not observed in the above animals.

  Immunization is carried out by administering the antigen subcutaneously or intravenously or intraperitoneally to the animal together with an appropriate adjuvant (for example, Freund's Adjuvant, aluminum hydroxide gel and pertussis vaccine, etc.). When the antigen is a partial peptide, a conjugate with a carrier protein such as BSA (bovine serum albumin) or KLH (Keyhole Limpet Hemocyanin) is prepared and used as an immunogen.

  The antigen is administered 5 to 10 times every 1 to 2 weeks after the first administration. Three to seven days after each administration, blood is collected from the fundus venous plexus, and the serum reacts with the antigen by using an enzyme immunoassay (Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory, 1988). A mouse, rat or hamster whose serum showed a sufficient antibody titer against the antigen used for immunization is provided as a source of splenocytes.

When the spleen cells are used for fusion of myeloma cells, the spleen is removed from the immunized mouse, rat or hamster 3 to 7 days after the final administration of the antigen substance, and the spleen cells are collected. The spleen is shredded in MEM medium (manufactured by Nissui Pharmaceutical), loosened with tweezers, centrifuged (1200 rpm, 5 minutes), then the supernatant is discarded, and tris-ammonium chloride buffer (pH 7.65) is used. Treated for 1-2 minutes to remove red blood cells, washed 3 times with MEM medium and provided as fusion splenocytes.
(3) Preparation of myeloma cells As myeloma cells, cell lines obtained from mice are used. For example, 8-azaguanine resistant mice (derived from BALB / c) myeloma cell line P3-X63Ag8-U1 (P3-U1) (Current Topics in Microbiology and Immunology, 18: 1-7, 1978), P3-NS1 / 1- Ag41 (NS-1) (European J. Immunology, 6: 511-519,1976), SP2 / O-Ag14 (SP-2) (Nature, 276: 269-270,1978), P3-X63-Ag8653 (653 J. Immunology, 123: 1548-1550, 1979), P3-X63-Ag8 (X63) (Nature, 256: 495-497, 1975), etc. are used. These cell lines consist of 8-azaguanine medium (RPMI-1640 medium with glutamine (1.5 mM), 2-mercaptoethanol (5 × 10 ^-5 M), gentamicin (10 μg / mL) and fetal calf serum (FCS). Medium added with 8-azaguanine (15 μg / mL) to the added medium (hereinafter referred to as normal medium)], but subcultured to normal medium 3-4 days before cell fusion, Secure a cell count of 2 x 10 ⁷ or more.
(4) Cell fusion The above-mentioned antibody-producing cells and myeloma cells are thoroughly washed with MEM medium or PBS (1.83 g disodium phosphate, 0.21 g monopotassium phosphate, 7.65 g sodium chloride, 1 L distilled water, pH 7.2). After mixing the cells so that the number of cells is antibody-producing cells: myeloma cells = 5 to 10: 1 and centrifuging (1,200 rpm, 5 minutes), the supernatant was discarded and the precipitated cells were thoroughly loosened. Then, at 37 ° C. with stirring, 2 g of polyethylene glycol-1,000 (PEG-1,000), 2 mL of MEM and 0.7 mL of dimethyl sulfoxide were added at a volume of 0.2 to 1 mL per 10 ⁸ antibody-producing cells, and the mixture was added for 1 to 2 minutes Add 1-2 ml of MEM medium several times each time, and then add MEM medium to a total volume of 50 mL. After centrifugation (900 rpm, 5 minutes), discard the supernatant, loosen the cells gently, suck with a pipette, blow gently to remove the cells into the HAT medium [normal medium with hypoxanthine (10 ^-4 M), thymidine (Medium ^{supplemented with} (1.5 × 10 ⁻⁵ M) and aminopterin (4 × 10 ⁻⁷ M)) Suspend in 100 mL. This suspension is dispensed into a 96-well culture plate at 100 μL / well and cultured at 37 ° C. for 7 to 14 days in a 5% CO ₂ incubator.

After culturing, a portion of the culture supernatant is taken, and a monoclonal antibody reactive to any of HB-EGF, secreted HB-EGF, and membrane HB-EGF bound to the cell membrane by a binding assay described later Select hybridomas or purified antibodies that produce
Next, cloning was repeated twice by the limiting dilution method (the first time was HT medium (the medium obtained by removing aminopterin from the HAT medium), and the second time was using a normal medium). Those found are selected as monoclonal antibody-producing hybridoma strains.
(5) Preparation of monoclonal antibody To 8-10 week-old mice or nude mice treated with pristane [0.5 mL of 2,6,10,14-tetramethylpentadecane (Pristane) was intraperitoneally administered and reared for 2 weeks] The anti-HB-EGF monoclonal antibody-producing hybridoma cells obtained in (4) are injected intraperitoneally 2 × 10 ^{6 to} 5 × 10 ⁷ cells / mouse. The hybridoma becomes ascites tumor in 10-21 days. Ascites was collected from this mouse, centrifuged (3,000 rpm, 5 minutes) to remove solids, salted out with 40-50% ammonium sulfate, caprylic acid precipitation method, DEAE-Sepharose column, protein A column Alternatively, purification using a gel filtration column is performed, and IgG or IgM fractions are collected and used as a purified monoclonal antibody.

The subclass of the antibody is determined by an enzyme immunoassay using a subcluster epiting kit. The amount of protein is calculated from the Raleigh method and absorbance at 280 nm.
(6) Binding assay An antigen is obtained by introducing an expression vector containing cDNA encoding HB-EGF used in the present invention into Escherichia coli, yeast, insect cells, animal cells, etc. by the method described in (1). In addition, purified HB-EGF or partial peptides obtained from transgenic cells, recombinant proteins, or human tissues are used. When the antigen is a partial peptide, a conjugate with a carrier protein such as BSA (bovine serum albumin) or KLH (Keyhole Limpet Hemocyanin) is prepared and used.

  Among these antigens, secreted HB-EGF and cell lines in which HB-EGF are bound to the cells are dispensed into 96-well plates and solidified, and then used as the first antibody as immunized animal serum, monoclonal antibody The culture supernatant or purified antibody of the hybridoma that produces is reacted. After thoroughly washing with PBS or PBS-0.05% Tween, an anti-immunoglobulin antibody labeled with biotin, an enzyme, a chemiluminescent substance, a radiation compound or the like is dispensed and reacted as a second antibody. After thoroughly washing with PBS-Tween, the reaction is carried out according to the labeling substance of the second antibody.

A hybridoma or purified antibody that produces a monoclonal antibody reactive to any of HB-EGF, secretory HB-EGF, and membrane HB-EGF bound to the cell membrane can be selected by the method described above. it can.
Among the obtained monoclonal antibodies, antibodies having binding inhibitory activity of secreted HB-EGF to HB-EGF receptor include monoclonal antibody KM3566 produced by hybridoma cell line KM3566 and monoclonal antibody produced by hybridoma cell line KM3567. Examples include monoclonal antibodies produced by KM3567 and hybridoma KM3579. The hybridoma cell line KM3579 was founded on January 24, 2006 in accordance with the Budapest Treaty at the FERM BP- Deposited as 10491.
Furthermore, whether or not the obtained monoclonal antibody has neutralizing activity against secretory HB-EGF can be confirmed by performing a cell growth inhibition assay using HB-EGF-dependent cells. .

The cell used in the cell growth inhibition assay may be any cell as long as it has a receptor capable of binding to secretory HB-EGF. Specifically, the EGF receptor gene is a mouse myeloid cell line. Examples include cell lines obtained by introduction into 32D clone3 (ATCC No. CRL-11346).
The obtained monoclonal antibody and secreted HB-EGF are reacted on a plate, and then the above-described cell line is added and cultured. As a control, the above-mentioned cell line is similarly added to a plate to which secretory HB-EGF is added and no monoclonal antibody is added, and a plate to which neither monoclonal antibody nor secretory HB-EGF is added. By measuring the number of cells in each plate, the cell growth inhibition rate can be determined. A monoclonal antibody having a high cell growth inhibition rate can be selected as a monoclonal antibody having neutralizing activity.

  Specific examples of the monoclonal antibody having neutralizing activity of the present invention include monoclonal antibody KM3566 produced by hybridoma cell line KM3566 and monoclonal antibody KM3567 produced by hybridoma cell line KM3567. The hybridoma cell lines KM3566 and KM3567 were established on January 24, 2006 and March 23, 2006, respectively, based on the Budapest Treaty, National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (Higashi 1-chome, Tsukuba City, Ibaraki, Japan) It is deposited as FERM BP-10490 and FERM BP-10573 at No. 1 Center 6).

2. Production of human chimeric antibody and humanized antibody
(1) Construction of humanized antibody expression vector Any humanized antibody expression vector may be used as long as it is an expression vector for animal cells into which a gene encoding CH and / or CL of a human antibody is incorporated. Humanized antibody expression vectors can be constructed by cloning genes encoding human antibodies CH and CL, respectively, into animal cell expression vectors.

  The C region of a human antibody can be CH and CL of any human antibody, for example, the C region of the IgG1 subclass of the H chain of the human antibody (hereinafter referred to as hCγ1) and the κ class of the L chain of the human antibody C region (hereinafter referred to as hCκ). As genes encoding CH and CL of human antibodies, chromosomal DNA consisting of exons and introns can be used, and cDNA can also be used.

Any expression vector for animal cells can be used as long as it can incorporate and express a gene encoding the C region of a human antibody. For example, pAGE107 (Cytotechnology, 3 , 133-140, 1990), pAGE103 (Journal of Biochemistry, 101 , 1307-1310, 1987), pHSG274 (Gene, 27 , 223-232, 1984), pKCR (Proceedings of the National Academy) of Sciences of the United States of America, 78 , 1527-1531, 1981), pSG1βd2-4 (Cytotechnology, 4 , 173-180, 1990). Promoters and enhancers used in animal cell expression vectors include SV40 early promoter and enhancer (Journal of Biochemistry, 101 , 1307-1310, 1987), Moloney murine leukemia virus LTR promoter and enhancer (Biochemical & Biophysical Research Communications, 149 , 960-968, 1987), an immunoglobulin heavy chain promoter (Cell, 41 , 479-487, 1985) and an enhancer (Cell, 33 , 717-728, 1983).

The human chimeric antibody and the humanized antibody expression vector may be of either the type in which the antibody H chain and L chain are on separate vectors, or the type on the same vector (hereinafter referred to as tandem type). It can be used, but the balance between the ease of constructing human chimeric antibodies and humanized antibody expression vectors, the ease of introduction into animal cells, and the expression levels of antibody H and L chains in animal cells is balanced. From these points, a tandem humanized antibody expression vector is preferred (Journal of Immunological Methods, 167 , 271-278, 1994). Examples of tandem humanized antibody expression vectors include pKANTEX93 (WO97 / 10354) and pEE18 (Hybridoma, 17 , 559-567, 1998).
(2) Obtaining cDNA encoding V region of non-human animal antibody and analysis of amino acid sequence Non-human animal antibody, for example, cDNA encoding mouse antibody VH and VL, is obtained as follows. be able to.

  MRNA is extracted from the hybridoma to synthesize cDNA. The synthesized cDNA is cloned into a vector such as a phage or a plasmid to prepare a cDNA library. A recombinant phage or recombinant plasmid having cDNA encoding VH and a recombinant phage or recombinant plasmid having cDNA encoding VL using the C region portion or V region portion of the mouse antibody as a probe from the library Are isolated respectively. The entire base sequence of VH and VL of the target mouse antibody on the recombinant phage or recombinant plasmid is determined, and the entire amino acid sequence of VH and VL is deduced from the base sequence.

Any animal other than humans can be used as long as it can produce a hybridoma, such as a mouse, rat, hamster, or rabbit.
Methods for preparing total RNA from hybridoma include guanidine thiocyanate-cesium trifluoroacetate method (Methods in Enzymology, 154 , 3-28, 1987), and methods for preparing mRNA from total RNA include oligo (dT). Examples thereof include an immobilized cellulose column method (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Lab. Press New York, 1989). Examples of kits for preparing mRNA from hybridomas include Fast Track mRNA Isolation Kit (Invitrogen) and Quick Prep mRNA Purification Kit (Pharmacia).

As cDNA synthesis and cDNA library preparation methods, conventional methods (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Lab. Press New York, 1989; Current Protocols in Molecular Biology, Supplement 1-34), or commercially available kits, For example, a method using Super Script ^™ Plasmid System for cDNA Synthesis and Plasmid Cloning (GIBCO BRL) or ZAP-cDNA Synthesis Kit (Stratagene) can be mentioned.

When preparing a cDNA library, any vector can be used as a vector into which cDNA synthesized using mRNA extracted from a hybridoma as a template can be incorporated. For example, ZAP Express (Strategies, 5 , 58-61, 1992), pBluescript II SK (+) (Nucleic Acids Research, 17 , 9494, 1989), λZAP II (Stratagene), λgt10, λgt11 (DNA Cloning: A Practical Approach, I, 49, 1985), Lambda BlueMid (Clontech), λExCell, pT7T3 18U (Pharmacia), pcD2 (Molecular & Cellular Biology, 3 , 280-289, 1983) and pUC18 (Gene, 33 , 103) -119, 1985) or the like.

Any Escherichia coli for introducing a cDNA library constructed by a phage or plasmid vector can be used as long as it can introduce, express and maintain the cDNA library. For example, XL1-Blue MRF '(Journal of Biotechnology, 23 , 271-289, 1992), C600 (Genetics, 59 , 177-190, 1968), Y1088, Y1090 (Science, 222 , 778-782, 1983), NM522 (Journal of Molecular Biology, 166 , 1-19, 1983), K802 (Journal of Molecular Biology, 16 , 118-133, 1966) and JM105 (Gene, 38 , 275-276, 1985) are used.

  The selection of cDNA clones encoding VH and VL of non-human animal antibodies from cDNA libraries includes colony hybridization or plaque hybridization (Molecular hybridization method using radioisotope or fluorescently labeled probe). Cloning: A Laboratory Manual, Cold Spring Harbor Lab. Press New York, 1989). In addition, primers were prepared, and cDNA or cDNA library synthesized from mRNA was used as a template for Polymerase Chain Reaction (hereinafter referred to as PCR method; Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Lab. Press New York, 1989; CDNAs encoding VH and VL can also be prepared by Current Protocols in Molecular Biology, Supplement 1-34).

The cDNA selected by the above method is cleaved with an appropriate restriction enzyme or the like, and then cloned into a plasmid vector such as pBluescript SK (-) (Stratagene), and a commonly used nucleotide sequence analysis method such as the dideoxy method (Proceedings) of the National Academy of Sciences of the United States of America, 74 , 5463-5467, 1977), etc., and analyzing the cDNA using an automatic base sequence analyzer ABI PRISM 377 (ABI). Can be determined.

  By deducing the total amino acid sequence of VH and VL from the determined nucleotide sequence and comparing it with the entire amino acid sequence of VH and VL of known antibodies (Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, 1991) It is possible to confirm whether the obtained cDNA encodes the complete amino acid sequence of VH and VL of the antibody including a signal sequence for secretion. Compare the complete amino acid sequence of the VH and VL of the antibody, including the signal sequence, with the entire amino acid sequence of the VH and VL of known antibodies (Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, 1991) Thus, the length of the signal sequence and the N-terminal amino acid sequence can be estimated, and further, the subgroup to which they belong can be known. Also, the amino acid sequences of CDRs of VH and VL can be found by comparing with the amino acid sequences of VH and VL of known antibodies (Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, 1991). it can.

Furthermore, using the complete amino acid sequence of VH and VL, any database such as BLAST method (Journal of Molecular Biology, 215 , 403-410, 1990) can be used for SWISS-PROT, PIR-Protein, etc. A homology search can be performed to examine the novelty of the sequence.
(3) Construction of human chimeric antibody expression vector VH and VL of antibody of non-human animal upstream of gene encoding human antibody CH and CL of humanized antibody expression vector described in 2 (1) above The cDNA encoding can be cloned to construct a human chimeric antibody expression vector. For example, a cDNA encoding VH and VL of a non-human animal antibody, a base sequence on the 3 ′ end side of the VH and VL of an antibody of a non-human animal, and a base sequence on the 5 ′ end side of CH and CL of the human antibody Each of which is linked to a synthetic DNA having a recognition sequence for an appropriate restriction enzyme at both ends, and each encodes the human antibody CH and CL of the humanized antibody expression vector described in 2 (1) above They can be cloned upstream of the gene so that they are expressed in an appropriate form, and a human chimeric antibody expression vector can be constructed. Also, cDNA encoding VH and VL by PCR using a plasmid containing cDNA encoding VH and VL of non-human animal antibody as a template and a primer having an appropriate restriction enzyme recognition sequence at the 5 'end. Are cloned so that they are expressed in an appropriate form upstream of the genes encoding the human antibody CH and CL of the humanized antibody expression vector described in 2 (1) above. Antibody expression vectors can be constructed.
(4) Construction of cDNA encoding V region of humanized antibody (CDR grafted antibody) cDNA encoding VH and VL of humanized antibody can be constructed as follows. First, the VH and VL CDR amino acid sequences of the human antibody to be grafted with the VH and VL CDR amino acid sequences of the target non-human animal antibody are selected. Any amino acid sequence derived from the human antibody can be used as the amino acid sequence of VH and VL of the human antibody. For example, human antibody VH and VL FR amino acid sequences registered in databases such as Protein Data Bank, human antibody VH and VL FR subgroup consensus amino acid sequences (Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, 1991). Among them, in order to produce a humanized antibody having sufficient activity, the amino acid sequence of FR of VH and VL of the antibody of the target non-human animal It is desirable to select an amino acid sequence having as high a homology as possible (at least 60% or more). Next, the VH and VL CDR amino acid sequences of the target non-human animal antibody are transplanted into the VH and VL FR amino acid sequences of the selected human antibody, and the VH and VL amino acid sequences of the humanized antibody are transferred. design. The designed amino acid sequence is converted into a base sequence in consideration of the frequency of use of codons found in the base sequence of the antibody gene (Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, 1991). A nucleotide sequence encoding the amino acid sequence of VH and VL is designed. Based on the designed base sequence, several synthetic DNAs with a length of about 150 bases are synthesized, and PCR is performed using them. In this case, it is preferable to design four synthetic DNAs for both VH and VL from the reaction efficiency in PCR and the length of DNA that can be synthesized.

In addition, by introducing an appropriate restriction enzyme recognition sequence into the 5 ′ end of the synthetic DNA located at both ends, it can be easily cloned into the humanized antibody expression vector constructed in 2 (1) above. After the PCR reaction, the amplified product is cloned into a plasmid such as pBluescript SK (-) (manufactured by Stratagene), the base sequence is determined by the method described in 2 (2) above, and the desired humanized antibody VH and VL are determined. A plasmid having a base sequence encoding the amino acid sequence is obtained.
(5) Modification of the amino acid sequence of the V region of a humanized antibody A humanized antibody can be obtained by transplanting only the VH and VL CDRs of the target non-human animal antibody to the VH and VL FRs of the human antibody. It is known that the antigen binding activity is reduced as compared with the antibody of the original non-human animal (BIO / TECHNOLOGY, 9 , 266-271, 1991). This is because, in the VH and VL of the original non-human animal antibody, not only CDR but also some amino acid residues of FR are directly or indirectly involved in antigen binding activity. Residues are thought to change to different amino acid residues of FRs of VH and VL of human antibodies with CDR grafting. In order to solve this problem, humanized antibodies interact directly with the amino acid residues involved in antigen binding and CDR amino acid residues in the VH and VL FR amino acid sequences of human antibodies. Or identify amino acid residues that are indirectly involved in antigen binding by maintaining the three-dimensional structure of the antibody, and changing them to amino acid residues found in the antibody of the original non-human animal. Increased antigen binding activity (BIO / TECHNOLOGY, 9 , 266-271, 1991). In the production of humanized antibodies, the most important point is how to efficiently identify the amino acid residues of FRs involved in these antigen-binding activities. For this reason, X-ray crystallography (Journal of Molecular Biology, 112 , 535-542, 1977) or computer modeling (Protein Engineering, 7 , 1501-1507, 1994), etc., the construction and analysis of the three-dimensional structure of antibodies has been performed. The information on the three-dimensional structure of these antibodies has provided a lot of useful information for the production of humanized antibodies, but on the other hand, methods for producing human CDR-grafted antibodies that can be applied to any antibody have not yet been established. However, at present, various trials and errors are required, such as preparing several types of variants for each antibody and examining the correlation with each antigen-binding activity.

Modification of amino acid residues of FR of human antibody VH and VL can be achieved by performing the PCR method described in 2 (4) above using synthetic DNA for modification. For the amplified product after PCR, the base sequence is determined by the method described in 2 (2) above, and it is confirmed that the target modification has been performed.
(6) Construction of humanized antibody expression vector Constructed in 2 (4) and (5) above the gene encoding the human antibody CH and CL of the humanized antibody expression vector described in 2 (1) above The cDNA encoding the humanized antibody VH and VL can be cloned to construct a humanized antibody expression vector. For example, among the synthetic DNAs used in the construction of humanized antibody VH and VL in 2 (4) and (5) above, appropriate restriction enzyme recognition sequences are introduced at the 5 ′ ends of the synthetic DNAs located at both ends. Thus, the humanized antibody expression vector described in 2 (1) above can be cloned so that they are expressed in an appropriate form upstream of the gene encoding the CH and CL of the human antibody.
(7) Transient expression of humanized antibodies In order to efficiently evaluate the antigen-binding activity of various types of humanized antibodies produced, the humanized antibody expression vectors described in 2 (3) and (6) above, Alternatively, transient expression of a humanized antibody can be performed using an expression vector obtained by modifying them. Any host cell that can express humanized antibodies can be used as a host cell into which an expression vector is introduced, but COS-7 cells (ATCC CRL1651) are generally used because of the high expression level. (Methods in Nucleic Acids Research, CRC press, 283, 1991). Methods for introducing expression vectors into COS-7 cells include the DEAE-dextran method (Methods in Nucleic Acids Research, CRC press, 283, 1991) and the lipofection method (Proceedings of the National Academy of Sciences of the United States of America, 84 , 7413-7417, 1987).

After the introduction of the expression vector, the expression level and antigen binding activity of the humanized antibody in the culture supernatant are measured by ELISA (Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory, Chapter 14, 1988; Monoclonal Antibodies: Principles and Practice, Academic Press Limited, 1996).
(8) Stable expression of humanized antibody By introducing the humanized antibody expression vector described in 2 (3) and (6) above into an appropriate host cell, a transformed cell that stably expresses the humanized antibody is obtained. Can do. Examples of the method for introducing an expression vector into a host cell include electroporation (Cytotechnology, 3 , 133-140, 1990). As the host cell into which the humanized antibody expression vector is introduced, any cell can be used so long as it can express the humanized antibody. For example, mouse SP2 / 0-Ag14 cells (ATCC CRL1581), mouse P3X63-Ag8.653 cells (ATCC CRL1580), CHO cells lacking dihydrofolate reductase gene (hereinafter referred to as dhfr ) (Proceedings of the National Academy of Sciences of the United States of America, 77 , 4216-4220, 1980), rat YB2 / 3HL.P2.G11.16Ag.20 cells (ATCC CRL1662, hereinafter referred to as YB2 / 0 cells) and the like.

  After the introduction of the expression vector, a transformant that stably expresses the humanized antibody is an animal cell culture containing a drug such as G418 sulfate (hereinafter referred to as G418) according to the method disclosed in JP-A-2-257891. It can be selected by culturing in a working medium. As an animal cell culture medium, RPMI1640 medium (manufactured by Nissui Pharmaceutical), GIT medium (manufactured by Nippon Pharmaceutical), EX-CELL302 medium (manufactured by JRH), IMDM (manufactured by GIBCO BRL), Hybridoma-SFM (GIBCO) BRL) or a medium obtained by adding various additives such as FBS to these mediums can be used. By culturing the obtained transformed cells in a medium, the humanized antibody can be expressed and accumulated in the culture supernatant. The expression level and antigen binding activity of the humanized antibody in the culture supernatant can be measured by ELISA. In addition, the transformed cells can increase the expression level of the humanized antibody using a DHFR amplification system or the like according to the method disclosed in JP-A-2-57891.

Humanized antibodies can be purified from the culture supernatant of transformed cells using a protein A column (Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Chapter 8, 1988; Monoclonal Antibodies: Principles and Practice, Academic Press Limited, 1996). In addition, a purification method usually used in protein purification can be used. For example, it can be purified by combining gel filtration, ion exchange chromatography and ultrafiltration. The molecular weight of the purified humanized antibody H chain, L chain, or whole antibody molecule is determined by polyacrylamide gel electrophoresis (hereinafter referred to as PAGE: Nature, 227 , 680-685, 1970) or Western blotting (Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Chapter 12, 1988; Monoclonal Antibodies: Principles and Practice, Academic Press Limited, 1996).
(9) Evaluation of binding activity between humanized antibody and antigen The binding activity between the humanized antibody and the antigen can be evaluated using the ELISA described above.

3. Production of Antibody Fragments Antibody fragments can be produced by genetic engineering techniques or protein chemistry techniques based on the antibodies described in 1 and 2 above.
Examples of the genetic engineering technique include a method of constructing a gene encoding the target antibody fragment, and expressing and purifying it using an appropriate host such as an animal cell, a plant cell, an insect cell, or E. coli.

Examples of protein chemistry techniques include site-specific cleavage and purification methods using proteolytic enzymes such as pepsin and papain.
A method for producing Fab, F (ab ′) ₂ , Fab ′, scFv, diabody, and dsFv as antibody fragments will be specifically described below.
(1) Fabrication of Fab
Fab can be proteochemically produced by treating IgG with the proteolytic enzyme papain. After papain treatment, if the original antibody is an IgG subclass with protein A binding properties, it can be separated from IgG molecules and Fc fragments by passing through a protein A column and recovered as a uniform Fab (Monoclonal Antibodies: Principles and Practice, third edition, 1995). For IgG subclass antibodies that do not have protein A binding, Fab can be recovered in fractions eluted at low salt concentrations by ion exchange chromatography (Monoclonal Antibodies: Principles and Practice, third edition, 1995). In terms of genetic engineering, Fab can be produced mostly using Escherichia coli, or using insect cells or animal cells. For example, DNA encoding the V region of the antibody described in 2 (2), 2 (4) and 2 (5) above can be cloned into a Fab expression vector to produce a Fab expression vector. Any Fab expression vector can be used as long as it can incorporate and express Fab DNA. Examples thereof include pIT106 (Science, 240 , 1041-1043, 1988). Fab expression vectors can be introduced into appropriate E. coli, and Fabs can be produced and accumulated in inclusion bodies or periplasm. Inclusion bodies can be made into active Fabs by the refolding method usually used for proteins, and when expressed in the periplasm, active Fabs leak into the culture supernatant. After refolding or from the culture supernatant, a uniform Fab can be purified by using a column to which an antigen is bound (Antibody Engineering, A Practical Guide, WH Freeman and Company, 1992).
(2) Preparation of F (ab ') ₂
F (ab ′) ₂ can be produced proteochemically by treating IgG with the proteolytic enzyme pepsin. After the treatment with pepsin, it can be recovered as uniform F (ab ′) ₂ by the same purification operation as Fab (Monoclonal Antibodies: Principles and Practice, third edition, Academic Press, 1995). Alternatively, Fab ′ described in the following 3 (3) may be treated with maleimide such as o-PDM or bismaleimide hexane to thioether bond, or DTNB [5,5′-dithiobis (2-nitrobenzoic acid)] It can also be produced by a method of treating with SS and bonding with SS (Antibody Engineering, A Practical Approach, IRL PRESS, 1996).
(3) Fabrication of Fab '
Fab ′ can be obtained by treating F (ab ′) ₂ described in 3 (2) above with a reducing agent such as dithiothreitol. In addition, Fab 'can be produced by genetic engineering using E. coli, insect cells, animal cells, and the like. For example, DNA encoding the V region of the antibody described in 2 (2), 2 (4) and 2 (5) above can be cloned into a Fab ′ expression vector to prepare a Fab ′ expression vector. As the Fab ′ expression vector, any vector can be used so long as it can incorporate and express Fab ′ DNA. An example is pAK19 (BIO / TECHNOLOGY, 10 , 163-167, 1992). Fab 'expression vectors can be introduced into appropriate E. coli and Fab' can be produced and accumulated in inclusion bodies or periplasm. Inclusion bodies can be made into active Fab 'by the refolding method usually used for proteins, and when expressed in the periplasm, treatments such as partial digestion with lysozyme, osmotic shock, sonication, etc. The bacteria can be crushed and recovered outside the cells. Uniform Fab 'can be purified after refolding or from the bacterial crush solution by using a protein G column or the like (Antibody Engineering, A Practical Approach, IRL PRESS, 1996).
(4) Production of scFv
scFv can be prepared by genetic engineering using phage or E. coli, insect cells, animal cells, and the like. For example, the scFv expression vector can be prepared by cloning a DNA encoding the V region of the antibody described in 2 (2), 2 (4) and 2 (5) above into an scFv expression vector. As the scFv expression vector, any vector can be used as long as it can incorporate and express scFv DNA. For example, pCANTAB5E (manufactured by Pharmacia), pHFA (Human Antibodies & Hybridomas, 5 , 48-56, 1994) and the like can be mentioned. By introducing the scFv expression vector into appropriate Escherichia coli and infecting the helper phage, a phage expressing scFv fused to the phage surface protein on the phage surface can be obtained. In addition, scFv can be produced and accumulated in inclusion bodies or periplasm of E. coli into which the scFv expression vector has been introduced. Inclusion bodies can be made into active scFv by the refolding method usually used for proteins, and when expressed in the periplasm, by partial digestion with lysozyme, osmotic shock, sonication, etc. The bacteria can be crushed and recovered outside the cells. Uniform scFv can be purified after refolding or from the bacterial disruption solution by using cation exchange chromatography (Antibody Engineering, A Practical Approach, IRL PRESS, 1996).
(5) Production of diabody
A diabody can be produced by genetic engineering, mostly using Escherichia coli, insect cells and animal cells. For example, a DNA in which VH and VL of the antibody described in 2 (2), 2 (4) and 2 (5) above are linked so that the amino acid residue encoded by the linker is 8 residues or less is prepared, and diabody A diabody expression vector can be prepared by cloning into an expression vector. Any diabody expression vector may be used as long as it can incorporate and express diabody DNA. For example, pCANTAB5E (manufactured by Pharmacia), pHFA (Human Antibodies Hybridomas, 5 , 48, 1994) and the like can be mentioned. Diabodies can be produced and accumulated in inclusion bodies or periplasm of E. coli introduced with a diabody expression vector. Inclusion bodies can be made into active diabodies by the refolding method normally used for proteins, and when expressed in the periplasm, they can be treated by partial digestion with lysozyme, osmotic shock, sonication, etc. The bacteria can be crushed and recovered outside the cells. Uniform diabody can be purified after refolding or from the crush solution of bacteria by using cation exchange chromatography (Antibody Engineering, A Practical Approach, IRL PRESS, 1996).
(6) Production of dsFv
In terms of genetic engineering, dsFv can be produced mostly using Escherichia coli, insect cells, animal cells, and the like. First, mutations were introduced at appropriate positions in the DNAs encoding VH and VL of the antibodies described in 2 (2), 2 (4) and 2 (5) above, and the encoded amino acid residues were substituted with cysteine. Make DNA. Each of the prepared DNAs can be cloned into a dsFv expression vector to prepare VH and VL expression vectors. As the dsFv expression vector, any vector can be used so long as it can incorporate and express dsFv DNA. An example is pULI9 (Protein Engineering, 7 , 697-704, 1994). VH and VL expression vectors can be introduced into appropriate E. coli, and dsFv can be produced and accumulated in inclusion bodies or periplasm. After obtaining and mixing VH and VL from inclusion bodies or periplasm, active dsFv can be obtained by a refolding method usually used for proteins. After refolding, it can be further purified by ion exchange chromatography and gel filtration (Protein Engineering, 7 , 697-704, 1994).

4). Medicament and therapeutic agent of the present invention A medicament containing the monoclonal antibody of the present invention as an active ingredient can treat various diseases involving HB-EGF.
Diseases involving HB-EGF include cancer, heart disease, arteriosclerosis and the like.
Examples of cancer include solid cancers such as breast cancer, liver cancer, pancreatic cancer, bladder cancer, ovarian cancer, and ovarian germ cell tumor. Also included are continuity, hematogenous or lymphatic metastasis associated with any solid cancer, metastatic cancer due to peritoneal dissemination, and the like. In addition, cancer types such as leukemia (acute myeloid leukemia, T-cell leukemia, etc.), lymphoma, hematopoietic cell-derived cancer such as myeloma (blood cancer, hematological cancer, blood cancer) can be mentioned.

The medicament comprising the antibody of the present invention or an antibody fragment thereof as an active ingredient is usually mixed with one or more pharmacologically acceptable carriers and produced by any method well known in the technical field of pharmaceutical sciences. It is desirable to provide it as a pharmaceutical preparation.
It is desirable to use the administration route most effective for treatment. In the case of an antibody or peptide preparation, intravenous administration is desirable. Examples of the dosage form include sprays, capsules, tablets, granules, syrups, emulsions, suppositories, injections, ointments, tapes and the like.

  Suitable formulations for oral administration include emulsions, syrups, capsules, tablets, powders, granules and the like. Liquid preparations such as emulsions and syrups include saccharides such as water, sucrose, sorbitol and fructose, glycols such as polyethylene glycol and propylene glycol, oils such as sesame oil, olive oil and soybean oil, p-hydroxybenzoic acid Preservatives such as esters, and flavors such as strawberry flavor and peppermint can be used as additives. Capsules, tablets, powders, granules, etc. are excipients such as lactose, glucose, sucrose, mannitol, disintegrants such as starch and sodium alginate, lubricants such as magnesium stearate and talc, polyvinyl alcohol, hydroxy A binder such as propylcellulose and gelatin, a surfactant such as fatty acid ester, a plasticizer such as glycerin and the like can be used as additives.

  Formulations suitable for parenteral administration include injections, suppositories, sprays and the like. The injection is prepared using a carrier made of a salt solution, a glucose solution, or a mixture of both. Suppositories are prepared using a carrier such as cacao butter, hydrogenated fat or carboxylic acid. The spray is prepared using a carrier that does not irritate the antibody or peptide itself or the recipient's oral cavity and airway mucosa and disperses the compound as fine particles to facilitate absorption. Specific examples of the carrier include lactose and glycerin. Depending on the nature of the antibody and the carrier used, preparations such as aerosols and dry powders are possible. In these parenteral preparations, the components exemplified as additives for oral preparations can also be added.

The dose or frequency of administration varies depending on the intended therapeutic effect, administration method, treatment period, age, body weight, etc., but is usually 10 μg / kg to 8 mg / kg per day for an adult.

The present invention will be described more specifically by the following examples. However, the examples are illustrative of the present invention and do not limit the scope of the present invention.

Preparation of anti-HB-EGF monoclonal antibody (1) Preparation of immunogen
Recombinant secreted human HB-EGF (catalog number 259-HE / CF) manufactured by R & D Systems was dissolved in Dulbecco's phosphate buffer (PBS) and used as an immunogen.
(2) Immunization of animals and preparation of antibody-producing cells 25 μg of the recombinant secretory human HB-EGF prepared in Example 1 (1) was added with aluminum hydroxide adjuvant [Antibodies-A Laboratory ManuaL, CoLd Spring Harbor Laboratory, p99, 1988]. HB-EGF-deficient mice with 2 mg and pertussis vaccine (Chiba Serum Research Laboratories) 1 × 10 ⁹ cells (provided by Laboratory for Cell Function, Osaka University), PNAS, VOL.100, NO.100, 3221 -3226, 2003). From 2 weeks after administration, only 25 μg of HB-EGF was administered once a week for a total of 4 times. Partial blood was collected from the fundus vein of the mouse, the serum antibody titer thereof was examined by the enzyme immunoassay shown below, and the spleen was excised 3 days after the final immunization from the mouse showing a sufficient antibody titer. The spleen was shredded in MEM (Minimum Essential Medium) medium (Nissui Pharmaceutical Co., Ltd.), loosened with tweezers, and centrifuged (1200 rpm, 5 minutes). Tris-ammonium chloride buffer (PH7.6) was added to the obtained precipitate fraction, and erythrocytes were removed by treatment for 1-2 minutes. The resulting precipitate fraction (cell fraction) was washed 3 times with MEM medium and used for cell fusion.
(3) Enzyme immunoassay (binding ELISA)
For the assay, the recombinant human HB-EGF of Example 1 (1) was dispensed at 0.5 μg / mL and 50 μL / well onto a 96-well ELISA plate (Greiner) and allowed to stand overnight at 4 ° C. for adsorption. Used. After washing the plate, 1% bovine serum albumin (BSA) -PBS was added at 50 μL / well and left at room temperature for 1 hour to block remaining active groups. After standing, 1% BSA-PBS was discarded, and 50 μL / well of immunized mouse antiserum and hybridoma culture supernatant were dispensed as primary antibodies to the plate and left for 2 hours. The plate was washed with 0.05% polyoxyethylene (20) sorbitan monolaurate [(ICI trademark Tween 20 equivalent: manufactured by Wako Pure Chemical Industries)] / PBS (hereinafter referred to as Tween-PBS) as a secondary antibody. 50 μL / well of peroxidase-labeled rabbit anti-mouse IgG gamma chain (Kirkegaard and Perry Laboratories) was added and left at room temperature for 1 hour. After washing the plate with Tween-PBS, ABTS [2.2-azinobis (3-ethylbenzothiazole-6-sulfonic acid) ammonium] substrate solution [1 mmoL / L ABTS / 0.1 moL / L citrate buffer (PH4.2) , 0.1% H ₂ O ₂ ] was added, the color was developed, and the absorbance at OD 415 nm was measured using a plate reader (Emax; Molecular Devices).
(4) Preparation of mouse myeloma cells
8-Azaguanine-resistant mouse myeloma cell line P3X63Ag8U.1 (P3-U1: purchased from ATCC) is cultured in 10% fetal bovine serum-supplemented RPMI1640 (Invitrogen), and 2 × 10 ⁷ or more cells are secured during cell fusion And used as a parent strain for cell fusion.
(5) Production of hybridoma The mouse spleen cells obtained in Example 1 (2) and the myeloma cells obtained in Example 1 (4) were mixed at 10: 1 and centrifuged (1200 rpm, 5 Minutes). Thoroughly disaggregate the cells of the resulting precipitate fraction, and mix with 10 ⁸ polyethylene glycol-1000 (PEG-1000) 1 g, MEM medium 1 mL, and dimethyl sulfoxide 0.35 mL at 37 ° C. with stirring. 0.5 mL per mouse spleen cell was added, and 1 mL of MEM medium was added several times to the suspension every 1 to 2 minutes, and then the MEM medium was added so that the total volume became 50 mL. After centrifuging the suspension (900 rpm, 5 minutes) and gently loosening the cells of the obtained precipitate fraction, the cells were gently removed by sucking and sucking with a pipette into HAT medium [10% fetal bovine serum. Suspended in 100 mL of a medium in which HAT Media Supplement (manufactured by Invitrogen) was added to RPMI1640 medium]. The suspension was dispensed at 200 μL / well into a 96-well culture plate and cultured at 37 ° C. for 10-14 days in a 5% CO ₂ incubator. After culturing, the culture supernatant is examined by the enzyme immunoassay described in Example 1 (3), a well that reacts with recombinant human HB-EGF is selected, and cloning by limiting dilution is repeated twice from the cells contained therein. Anti-HB-EGF monoclonal antibody producing hybridoma strains KM3566, KM3567 and KM3579 were established.
(6) Purification of monoclonal antibody 5-20 × 10 ⁶ cells / animal of the hybridoma strain obtained in Example 1 (5) were injected intraperitoneally into pristane-treated 8-week-old nude female mice (BALB / c). After 10 to 21 days, ascites was collected (1 to 8 mL / animal) from a mouse in which ascites accumulated due to the hybridoma becoming ascites tumor. The ascites was centrifuged (3000 rpm, 5 minutes) to remove solids. The purified IgG monoclonal antibody was obtained by purification by the caprylic acid precipitation method [Antibodies-A Laboratory ManuaL, Cold Spring Harbor Laboratory, 1988]. The subclass of the monoclonal antibody was determined by ELISA using a sub-clustering kit. The subclass of monoclonal antibody KM3566 was IgG1, the subclass of KM3567 was IgG1, and monoclonal antibody KM3579 was IgG2b.

Reactivity of anti-HB-EGF monoclonal antibody against HB-EGF (1) Reactivity with HB-EGF in binding ELISA This was performed according to the method shown in Example 1 (3). For the primary antibody, purified anti-HB-EGF monoclonal antibodies KM3566, KM3567, KM3579, commercially available anti-HB-EGF monoclonal antibody MAB259 (manufactured by R & D), and negative control antibody KM511 (anti-GCSF derivative monoclonal antibody), The product diluted stepwise from 5-g dilution from μg / mL was used. The results are shown in Fig. 1-1.

Anti-HB-EGF monoclonal antibodies KM3566, KM3567, KM3579, and MAB259 all reacted with recombinant human HB-EGF and did not react with BSA at all.
(2) Reactivity with HB-EGF in Western blot
For each lane, 20 ng of recombinant human HB-EGF (R & D) was fractionated by SDS-polyacrylamide electrophoresis, and the gel after electrophoresis was transferred to a PVDF membrane. After blocking the membrane with 10% BSA-PBS, the purified antibodies of anti-HB-EGF monoclonal antibodies KM3566, KM3567, KM3579, MAB259 and negative control antibody KM511 were each diluted to 1 μg / mL with 10% BSA-PBS. And allowed to react at room temperature for 2 hours. The membrane was thoroughly washed with Tween-PBS, and diluted peroxidase-labeled mouse immunoglobulin (Zymet) was reacted at room temperature for 1 hour. The membrane was thoroughly washed with Tween-PBS, and bands were detected using ECL ^™ western blotting detection reagents (Amersham Pharmacia).

Anti-HB-EGF monoclonal antibodies KM3566, KM3567, KM3579, and MAB259 all detected a band in the vicinity of 15-30 kilodalton (hereinafter referred to as kDa) corresponding to the molecular weight of recombinant secreted human HB-EGF.
(3) Evaluation of HB-EGF-EGFR binding inhibitory activity of anti-HB-EGF monoclonal antibody The anti-HB-EGF monoclonal antibodies KM3566, KM3567, KM3579 and MAB259 were labeled with 32D / EGFR cells and biotin. It examined using HB-EGF.

Recombinant secretory HB-EGF was labeled with biotin by EZ-Link Sulfo-NHS-Biotin (Pierce) by a conventional method.
KM3566, KM3567, KM3579 and MAB259 were diluted stepwise at a 5-fold dilution from 10 μg / mL and dispensed into a 96-well plate at 50 μL / well. Thereafter, 32D / EGFR cells were dispensed at 1 × 10 ⁴ cells / 50 μL / well. Furthermore, biotin-labeled HB-EGF and Alexa 647-labeled streptavidin diluted to an optimal concentration were dispensed at 10 μL / well and 50 μL / well, respectively, and mixed, and then allowed to react for 3 hours in the dark at room temperature. The wavelength of 650 nm to 685 nm excited by laser light 633 nm He / Ne was measured by 8200 Cellular Detection System (Applied Biosystems).

  As a result, as shown in FIG. 1-2, KM3566, KM3567, KM3579, and MAB259 all inhibited the binding of biotinylated HB-EGF to EGFR in an antibody concentration-dependent manner. Therefore, it was revealed that all anti-HB-EGF monoclonal antibodies inhibit the binding between HB-EGF and EGFR.

Examination of neutralizing activity of anti-HB-EGF monoclonal antibody against HB-EGF HB-EGF neutralizing activity of anti-HB-EGF monoclonal antibodies KM3566, KM3567, KM3579 and MAB259 was inhibited by cell growth using HB-EGF-dependent cells Investigated by assay. As an HB-EGF-dependent cell, a cell line (hereinafter referred to as 32D / EGFR) constructed by introducing the EGFR gene into a mouse bone marrow-derived cell line 32D clone3 (ATCC CRL-11346) was used. Purify the anti-HB-EGF monoclonal antibodies KM3566, KM3567, KM3579, MAB259, and the negative control antibody KM511 stepwise from 20 μg / mL at a 3- to 4-fold dilution, and distribute to a 96-well plate at 50 μL / well. Noted. Next, 0.1 μg / mL recombinant human HB-EGF (R & D) was dispensed at 10 μL / well, mixed, and then reacted on ice for 2 hours. Thereafter, 32D / EGFR cells were seeded at 1 × 10 ⁴ cells / 40 μL / well and cultured for 36 hours. Viable cell number measurement reagent SF (Nacalai Tesque) was added at 10 μL / well, and after 2 hours, the absorbance at OD450 nm was measured using a plate reader (Emax; Molecular Devices).

  Results of calculating the cell growth inhibition rate of each well with the absorbance of the wells with and without HB-EGF as 0% inhibition and the absorbance of the wells without and with HB-EGF as 100% inhibition Is shown in FIG. As a result, KM3566 showed the same inhibitory activity on HB-EGF-dependent growth as MAB259 against the cell line 32D / EGFR. Therefore, it was revealed that the two antibodies have the same level of HB-EGF neutralizing activity. On the other hand, KM3579 did not inhibit the growth of the cell line 32D / EGFR at all, and thus it was revealed that KM3579 has no HB-EGF neutralizing activity.

  Further, the results of examining the neutralizing activity of KM3567 in the same manner are shown in FIG. As a result, KM3567 had an activity of inhibiting HB-EGF-dependent cell proliferation, although its activity was weaker than that of KM3566.

Reactivity of anti-HB-EGF monoclonal antibody to membrane HB-EGF
1-5 × 10 ⁵ cells of human gastric cancer cell line MKN-28 (HSRRB JCRB0253), human ovarian cancer ES-2 (ATCC CRL-1978) and human breast cancer cell line MDA-MB-231 (ATCC HTB-26), Anti-HB-EGF monoclonal antibodies KM3566, KM3567, KM3579, MAB259 and negative control antibody KM511 diluted with 0.1% BSA-PBS to various concentrations were added and mixed to make a total volume of 50 μL. These cell suspensions were reacted for 40 minutes on ice and then washed three times with 0.1% BSA-PBS. 50 μL of FITC-labeled goat anti-mouse IgG + IgM (H + L) polyclonal antibody (Kirkegaard & Perry Laboratories) diluted with 0.1% BSA-PBS was added to the cells, and reacted for 40 minutes under ice cooling. After washing with 0.1% BSA-PBS, it was suspended in 0.1% BSA-PBS, and the fluorescence intensity was measured using a flow cytometer (Coulter).

  FIG. 3 shows the mean fluorescence intensity (MFI value) when MKN-28 and ES-2 were reacted with the above monoclonal antibody at a 2-fold dilution from 20 μg / mL. FIG. 4 shows a histogram when the above-described monoclonal antibody is reacted with human breast cancer cell line MDA-MB-231 at 20 μg / mL. As a result, binding activity of KM3566 and KM3579 was observed for MKN-28, and binding activity was observed in the order of KM3566> KM3579 for ES-2. Furthermore, in MDA-MB-231, binding activity was observed in the order of KM3566> KM3567≈KM3579. In any cell, the MFI value of MAB259 was similar to that of the negative control antibody KM511 and the non-antibody added negative control, and binding to the cell was hardly observed. From the above, it has been clarified that the monoclonal antibodies KM3566, KM3567 and KM3579 bind to the membrane type of the cancer cell line and HB-EGF bound to the cell membrane.

Isolation and analysis of cDNA encoding variable region of anti-HB-EGF monoclonal antibody (1) Preparation of mRNA from anti-HB-EGF monoclonal antibody-producing hybridoma cells From the hybridoma KM3566 described in Example 1, RNAeasy Maxi kit (QIAGEN ) And OligotexTM-dT30 <Super> mRNA Purification Kit (Takara) and about 4.8 μg of mRNA was prepared from 5 × 10 ⁷ hybridoma cells according to the attached instruction manual.
(2) Gene cloning of the H chain and L chain variable regions of anti-HB-EGF monoclonal antibody KM3566 From 1 μg of the mRNA of anti-HB-EGF monoclonal antibody KM3566 obtained in Example 5 (1), BD SMARTTM RACE cDNA Amplification Kit ( Using BD Biosciences), a cDNA having the BD SMART II ™ A Oligonucleotide sequence attached to the kit on the 5 ′ side was obtained according to the attached instruction manual. Using the cDNA as a template, a PCR reaction was performed using the universal primer Amix attached to the kit and a mouse Ig (γ) -specific primer having the nucleotide sequence represented by SEQ ID NO: 6 to amplify the VH cDNA fragment. PCR was performed using a mouse Ig (κ) -specific primer having the nucleotide sequence represented by SEQ ID NO: 7 in place of the Ig (γ) -specific primer to amplify a VL cDNA fragment. PCR is heated at 94 ° C for 5 minutes, followed by 5 reaction cycles of 94 ° C for 30 seconds and 72 ° C for 3 minutes, 94 ° C for 30 seconds, 70 ° C for 30 seconds, 72 ° C for 3 minutes After 30 cycles of 5 cycles, 94 ° C. for 30 seconds, 68 ° C. for 30 seconds, and 72 ° C. for 3 minutes, each was reacted at 72 ° C. for 10 minutes. PCR was performed using PTC-200 DNA Engine (manufactured by BioRad).

In order to clone the obtained PCR product and determine the base sequence, it was separated by agarose gel electrophoresis, and the PCR product of about 600 bp each of the H chain and the L chain was extracted using Gel Extraction Kit (manufactured by QIAGEN). The obtained extracted fragment was ligated to pBluescriptII SK (−) vector digested with SmaI using Ligation high (manufactured by Toyobo Co., Ltd.), followed by the method of Cohen et al. [Proc. Natl. Acad. Sci. USA, 69 , 2110 (1972)] was transformed into E. coli strain DH5α. The plasmid was extracted from the obtained transformant using an automatic plasmid extractor (Kurabo), and reacted according to the attached instructions using the BigDye Terminator Cycle Sequencing FS Ready Reaction Kit (PE Biosystems). The nucleotide sequence of the PCR product cloned by the sequencer ABI PRISM3700 was analyzed. As a result, a plasmid KM3566VH10G2 containing a full-length H chain cDNA and a plasmid KM3566VL10K2 containing an L chain cDNA having an ATG sequence presumed to be the initiation codon at the 5 ′ end of the cDNA were obtained.
(3) Analysis of amino acid sequence of V region of anti-HB-EGF monoclonal antibody The entire base sequence of VH contained in plasmid KM3566VH10G2 is SEQ ID NO: 8, and all amino acids of VH including the signal sequence deduced from this sequence The sequence is shown in SEQ ID NO: 9, the entire nucleotide sequence of the VL contained in the plasmid KM3566VL10K2 is shown in SEQ ID NO: 10, and the entire amino acid sequence of the VL including the signal sequence estimated from the sequence is shown in SEQ ID NO: 11, respectively. . Comparison with known mouse antibody sequence data [SEQUENCES of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)] and purified anti-HB-EGF monoclonal antibody KM3566 N-terminal amino acids of H and L chains From the comparison with the result of analyzing the sequence using a protein sequencer (manufactured by Shimadzu Corporation: PPSQ-10), each isolated cDNA is a full-length cDNA encoding anti-HB-EGF monoclonal antibody KM3566 containing a secretory signal sequence. It is clear that the 1st to 19th amino acid sequence described in SEQ ID NO: 9 for the H chain and the 1st to 20th amino acid sequence described in SEQ ID NO: 11 for the L chain are secretory signal sequences. It was.

  Next, the novelty of the amino acid sequence of VH and VL of the anti-HB-EGF monoclonal antibody KM3566 was examined. GCG Package (version 9.1, manufactured by Genetics Computer Group) was used as a sequence analysis system, and an amino acid sequence database of existing proteins was searched by the BLASTP method [Nucleic Acids Res., 25, 3389 (1997)]. As a result, VH and VL did not show completely identical amino acid sequences, and it was confirmed that VH and VL of anti-HB-EGF monoclonal antibody KM3566 have novel amino acid sequences.

  Further, CDRs of VH and VL of anti-HB-EGF monoclonal antibody KM3566 were identified by comparing with amino acid sequences of known antibodies. The amino acid sequences of CDR1, CDR2, and CDR3 of VH of anti-HB-EGF monoclonal antibody KM3566 are shown in SEQ ID NOs: 12, 13, and 14, and the amino acid sequences of CDR1, CDR2, and CDR3 of VL are shown in SEQ ID NOs: 15, 16, and 17, respectively. .

Preparation of anti-HB-EGF chimeric antibody (1) Construction of anti-HB-EGF chimeric antibody expression vector pKANTEX3566
Using the humanized antibody expression vector pKANTEX93 described in WO97 / 10354 and the plasmids KM3566VH10G2 and KM3566VL10K2 obtained in Example 5 (2), an anti-HB-EGF chimeric antibody expression vector pKANTEX3566 was constructed as follows. .

Using 100 ng of plasmid KM3566VH10G2 as a template, 10 μL of 10 × KOD buffer, 10 μL of 2 mmol / L dNTP, 2 μL of 25 mmol / L magnesium chloride, and 1 μL of primers each having a base sequence described in SEQ ID NOs: 18 and 19 of 10 μmol / L , KOD polymerase (manufactured by Toyobo Co., Ltd.) 1 μL, a total volume of 100 μL, heated at 96 ° C. for 3 minutes, followed by 25 cycles of 94 ° C. for 1 minute, 55 ° C. for 1 minute, and 72 ° C. for 1 minute And allowed to react at 72 ° C. for 8 minutes. By this reaction, a gene sequence encoding VH of KM3566 to which a restriction enzyme recognition sequence for insertion into pKANTEX93 was added was synthesized. Similarly, a primer having plasmid KM3566VL10K2 as a template, 100 ng, 10 × KOD buffer solution 10 μL, 2 mmol / L dNTP 10 μL, 25 mmol / L magnesium chloride 2 μL, 10 μmol / L of the nucleotide sequence described in SEQ ID NOs: 20 and 21 1 μL of each and 1 μL of KOD polymerase (Toyobo Co., Ltd.), a total volume of 100 μL, heated at 96 ° C. for 3 minutes, then 94 ° C. for 1 minute, 55 ° C. for 1 minute, 72 ° C. for 1 minute Was reacted for 8 minutes at 72 ° C. for 25 cycles. By this reaction, a gene sequence encoding VL of KM3566 to which a restriction enzyme recognition sequence for insertion into pKANTEX93 was added was synthesized. Each PCR reaction product is purified by ethanol precipitation, concentrated, and cloned into pBluescriptII SK (-) vector digested with SmaI, thereby encoding plasmid pKM3566VH containing the gene sequence encoding VH of KM3566 and VL Plasmid pKM3566VL containing the gene sequence was obtained. Next, to the vector pKANTEX93 and pKM3566VL obtained above, after adding the restriction enzyme BsiWI (New England Biolabs) and reacting at 55 ° C. for 1 hour, the restriction enzyme EcoRI (TaKaRa) was subsequently added, The reaction was carried out at 37 ° C for 1 hour. The reaction solution was subjected to agarose gel electrophoresis, and an about 12.8 kb EcoRI-BsiWI fragment of pKANTEX93 and an about 0.43 kb EcoRI-BsiWI fragment were collected using a QIAquick Gel Extraction Kit (manufactured by QIAGEN). . Ligation high (manufactured by Toyobo Co., Ltd.) was ligated with the two obtained fragments according to the attached instructions, and the resulting recombinant plasmid DNA solution was used to transform Escherichia coli DH5α (manufactured by Toyobo Co., Ltd.) did. Each plasmid DNA was prepared from a clone of the transformant and confirmed by restriction enzyme treatment to obtain a plasmid pKANTEX3566VL into which the target EcoRI-BsiWI fragment of about 0.43 kb was inserted. Next, the restriction enzyme ApaI (TaKaRa) was added to the pKANTEX3566VL and pKM3566VH obtained above and reacted at 37 ° C. for 1 hour, and then the restriction enzyme NotI (New England Biolabs) was added. The reaction was carried out at 1 ° C. for 1 hour. The reaction solution was fractionated by agarose gel electrophoresis, and ApaI-NotI fragments of about 13.2 kb pKANTEX3566VL and about 0.47 kb VH were recovered. The two fragments obtained were ligated using Ligation High (Toyobo Co., Ltd.) according to the attached instructions, and the resulting recombinant plasmid DNA solution was used to transform Escherichia coli DH5α strain (Toyobo Co., Ltd.). did. Each plasmid DNA was prepared from the clone of the transformant and confirmed by restriction enzyme treatment to obtain the plasmid pKANTEX3566 into which the target 0.47 kb ApaI-NotI fragment was inserted. The plasmid was reacted using the BigDye Terminator Cycle Sequencing FS Ready Reaction Kit (manufactured by PE Biosystems) according to the attached instruction, and the nucleotide sequence was analyzed with its sequencer ABI PRISM3700. As a result, an anti-HB-EGF chimeric antibody expression vector pKANTEX3566 in which the cDNA encoding the target KM3566 VH and the cDNA encoding VL were respectively cloned was obtained. A schematic diagram of vector construction is shown in FIG.
(2) Expression of anti-HB-EGF chimeric antibody in animal cells Using the anti-HB-EGF chimeric antibody expression vector pKANTEX3566 obtained in (1) above, The method [Antibody Engineering, A Practical Guide, WHFreeman and Company (1992)] was carried out to obtain a transformed strain KM3966 producing an anti-HB-EGF chimeric antibody.
(3) Acquisition of purified chimeric antibody After culturing the transformed strain KM3966 obtained in (2) above by the usual culture method, the cell suspension is recovered and centrifuged for 10 minutes at 3000 rpm and 4 ° C. After separation and collection of the culture supernatant, the solution was sterilized by filtration through a 0.22 μm pore size Millex GV filter (manufactured by Millipore). The anti-HB-EGF chimeric antibody KM3966 was purified from the obtained culture supernatant using a Protein A High-capacity resin (Milipore) column according to the attached instructions. The purity of the purified preparation of the anti-HB-EGF chimeric antibody KM3966 and the expressed molecular size were determined by SDS-PAGE using a gradient gel (ATTO, E-T520L) according to the attached instructions. confirmed.

  The results are shown in FIG. In the purified anti-HB-EGF chimeric antibody KM3966, one band was observed at a molecular weight of about 150 to 200 kDa under non-reducing conditions, and two bands of about 50 kDa and about 25 kDa were observed under reducing conditions. These molecular weights are about 150 kDa for IgG class antibodies under non-reducing conditions. Under reducing conditions, the SS bond in the molecule is cleaved, and a heavy chain with a molecular weight of about 50 kDa and a molecular weight of about 25 kDa. (Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory, Chapter 14 (1988), Monoclonal Antibodies-Principles and Practice, Academic Press Limited (1996)). Therefore, it was confirmed that the anti-HB-EGF chimeric antibody KM3966 was expressed as an antibody molecule with the correct structure.

Activity evaluation of anti-HB-EGF chimeric antibody (1) Binding activity to human solid cancer cell line In order to evaluate the binding of anti-HB-EGF chimeric antibody KM3966 obtained in Example 6 to human solid cancer cell line, a fluorescent antibody The following was examined by the law.
Human ovarian cancer cell lines MCAS (JCRB0240), RMG-I (JCRB IF050315), ES-2 (CRL1978), human breast cancer cell lines MDA-MB-231 (ATCC HTB-26), T47D (HTB-133), Various cell lines of SK-BR-3 (ATCC HTB-30), ZR-75-1 (ATCC CRL-1500), and human gastric cancer cell line MKN-28 (HSRRB JCRB0253) were combined with 0.02% -EDTA Solution (Nacalai Tesque). And then washed with PBS, and dispensed into a 96-well U-bottom plate (manufactured by FALCON) in an amount of 1-2 × 10 ⁵ pieces / 50 μL / well. An anti-HB-EGF chimeric antibody KM3966 solution prepared with 1% BSA-PBS at 20 μg / mL was dispensed at 50 μL / well, stirred with a plate mixer, and allowed to stand on ice for 30 minutes. After washing twice with PBS, the secondary antibody FITC-conjugated AffiniPure F (ab ') 2 Fragment Rabbit Anti-Human IgG (H + L) (Jackson Laboratories) diluted 100 times was added to each 50 μL / well, The mixture was stirred with a plate mixer and kept on ice for 30 minutes while being protected from light. After washing twice with PBS, the fluorescence intensity was measured using a flow cytometer EPICS XL System II v3.0 (manufactured by BECKMAN COULTER). As a negative control antibody, anti-FGF8 chimeric antibody KM3034 (US2004-0253234) was used.

The results are shown in FIG. Anti-HB-EGF chimeric antibody KM3966 bound to the membrane type of any human solid cancer cell line and HB-EGF bound to the cell membrane.
(2) Measurement of binding activity of anti-HB-EGF chimeric antibody KM3966 to human HB-EGF To analyze the binding activity of mouse antibody KM3566 and chimeric antibody KM3966 to human HB-EGF in terms of reaction kinetics, binding activity using Biacore Measurements were made. The following operations were all performed using Biacore T-100 (Biacore). Human HB-EGF (R & D) prepared to 5μg / mL using HBS-EP Buffer (Biacore) becomes 80RU (resonance unit) on CM5 sensor chip (Biacore) by amine coupling method It was solidified as follows. Thereafter, various antibodies diluted from 9 nmol / L to 5 levels are flowed on the chip at a speed of 10 μL / min, sensorgrams at each concentration are analyzed, and binding rate constants and dissociation rate constants of each antibody for human HB-EGF are calculated. did.

As a result, it was revealed that almost no dissociation reaction was observed after binding to human HB-EGF in both antibody concentrations, and the dissociation rate constant could not be calculated. On the other hand, the binding rate constant can be calculated, and the results are shown in Table 1. From this result, it was confirmed that both antibodies have substantially the same binding activity to human HB-EGF.
Table 1
Antibody Ka (1 / Ms)
KM3566 2.7 × 10 ⁵
KM3966 2.4 × 10 ⁵
(3) Reactivity of anti-HB-EGF monoclonal antibody against HB-EGF bound to the cell membrane Peel off the cell line with 0.02% -EDTA Solution (Nacalai Tesque), wash with PBS, and then RPMI1640 medium (GIBCO-BRL The supernatant was removed by centrifugation at 300 G for 5 minutes. Recombinant human HB-EGF (R & D) diluted with 0.1% BSA-PBS was added to the cells at 1 μg / mL and allowed to react at 37 ° C. for 10 minutes. When recombinant human HB-EGF was not added, only 0.1% BSA-PBS was added and reacted at 37 ° C. for 10 minutes in the same manner. After washing twice with 1% BSA-PBS, the anti-HB-EGF chimeric antibody KM3966 solution prepared to 10 μg / mL with 1% BSA-PBS is dispensed at 50 μL / well, stirred with a plate mixer, and placed on ice. Let sit for a minute. After washing twice with PBS, the secondary antibody FITC-conjugated AffiniPure F (ab ') ₂ diluted 100-fold was added to each plate at 50 μL / well, and Fragment Rabbit Anti-Human IgG (H + L) (Jackson Laboratories) was added. The mixture was agitated with a mixer, protected from light, and allowed to stand on ice for 30 minutes. After washing twice with PBS, the fluorescence intensity was measured with a flow cytometer EPICS XL System II v3.0 (manufactured by BECKMAN COULTER). An anti-FGF8 chimeric antibody (US2004-0253234) was used as a negative control antibody.

As a result, the reactivity of the anti-HB-EGF chimeric antibody KM3966 increased in the cells treated with recombinant HB-EGF in all cell lines compared to the cells not treated (FIG. 8). Therefore, it was revealed that the anti-HB-EGF chimeric antibody KM3966 of the present invention binds to both membrane type and HB-EGF bound to the cell membrane.
(4) Neutralizing activity against human solid cancer cell line In order to evaluate the neutralizing activity against HB-EGF of anti-HB-EGF chimeric antibody KM3966 obtained in Example 6, the HB-EGF-dependent growth inhibitory activity was measured. did. As HB-EGF-dependent cells, HB-EGF-positive human ovarian cancer cell line RMG-I (JCRB IF050315) and human gastric cancer cell line MKN-28 (HSRRB JCRB) were used.

Peel off the cell line with 0.02% -EDTA Solution (Nacalai Tesque), wash with PBS, add RPMI1640 medium (GIBCO-BRL) (serum-free), centrifuge at 300G for 5 minutes to remove the supernatant did. After suspending the cells in the same medium, RMG-I was seeded in a 96-well plate at 2.5 × 10 ³ cells / 50 μL / well and MKN-28 at 1 × 10 ⁴ cells / 50 μL / well. Recombinant human HB-EGF (R & D) diluted with 0.1% BSA-PBS, with a concentration of 3 ng / mL for RMG-I, 50 μL / well, and a concentration of 30 ng / mL for MKN-28 Were added at 50 μL / well, and anti-HB-EGF chimeric antibody KM3966 was diluted 10-fold from 30 μg / mL into 4 stages, and added at 50 μL / well and mixed. Human IgG (manufactured by Mitsubishi Pharma) was used as a negative control antibody. After culturing at 37 ° C. for 72 hours, add 15 μL / well of live cell counting reagent WST-1 (manufactured by Nacalai Tesque), and after 2 hours, use a plate reader (Emax; manufactured by MoLecular Devices) to measure the absorbance at OD450 nm. Measured.

The results are shown in FIG. Both RMG-I and MKN-28 showed cell growth by adding HB-EGF, and showed HB-EGF-dependent growth. The anti-HB-EGF chimeric antibody KM3966 inhibited HB-EGF-dependent cell proliferation in an antibody concentration-dependent manner and showed neutralizing activity.
(5) Antibody-dependent cytotoxic activity (ADCC activity)
The ADCC activity of the anti-HB-EGF chimeric antibody KM3966 obtained in Example 6 was measured according to the method shown below.
(5) -1 Preparation of target cell solution MCAS, RMG-I, ES-2 of human ovarian cancer cell lines, MDA-MB-231, T47D, SK-BR-3, ZR-75-1 of human breast cancer cell lines Human gastric cancer cell line MKN-28 cell lines were peeled with 0.02% -EDTA Solution (Nacalai Tesque) and phenol red-free RPMI1640 medium (Invitrogen) containing 1% FCS (JRH) After washing with (hereinafter referred to as ADCC medium), an optimal concentration was prepared with the same medium to obtain a target cell solution.
(5) -2 Preparation of effector cell solution Peripheral blood mononuclear cells (PBMCs) were separated from peripheral blood of healthy persons by the method described below. 50 mL of healthy human peripheral blood was collected with a syringe containing a small amount of heparin sodium injection N “Shimizu” (manufactured by Shimizu Pharmaceutical Co., Ltd.). The collected peripheral blood was diluted with the same amount of physiological saline (manufacturer name) and stirred well. On Polymorphprep (manufactured by NYCOMED) dispensed approximately 6.5 mL into a 15 mL tube (manufactured by Greiner), the same amount of diluted peripheral blood is gently layered, and centrifuged at room temperature for 800 G for 30 minutes to mononuclear cells. The layers were separated. After washing twice with the ADCC medium, it was adjusted to the optimum concentration with the same medium to obtain an effector cell solution.
(5) -3 Measurement of ADCC activity In each well of a 96-well U-bottom plate (manufactured by FALCON), 50 μL of the antibody diluted solution was dispensed, and 50 μL of the target cell solution prepared in (4) -1 ( 4) 50 μL of the effector cell solution prepared in -2 was added (the ratio of the effector cell (E) to the target cell (T) was 25), the total amount was 150 μL, and the mixture was reacted at 37 ° C. for 4 hours. Target cell spontaneous release values were obtained by adding 50 μL of target cell solution and 100 μL of medium, and target cell and effector cell spontaneous release values were obtained by adding 50 μL of target cell solution, 50 μL of effector cells and 50 μL of medium. . The target cell total release value was obtained by adding 50 μL of target cell solution and 80 μL of medium, and adding 20 μL of 9% Triton X-100 solution 45 minutes before the end of the reaction. After the reaction, the plate was centrifuged, and the lactate dehydrogenase (LDH) activity in the supernatant was detected by measuring the absorbance using LDH-Cytotoxic Test (manufactured by Wako) according to the attached instructions. ADCC activity was determined by the following formula.
(formula)
ADCC activity (%) = ([absorbance of specimen] − [absorbance of spontaneous release of target cells and effector cells]) / ([absorbance of total release of target cells] − [absorbance of spontaneous release of target cells]) × 100
The results are shown in FIG. The anti-HB-EGF chimeric antibody KM3966 exhibited cytotoxic activity against the HB-EGF-positive human solid cancer cell line in an antibody concentration-dependent manner.
(6) Antitumor activity evaluation using mouse xenograft
In order to evaluate the antitumor activity of the anti-HB-EGF chimeric antibody KM3966 obtained in Example 6, the evaluation was performed using human ovarian cancer, mouse xenograft early cancer of human breast cancer and advanced cancer model.
(6) -1 Evaluation in an early cancer model MCAS and ES-2 of human ovarian cancer cell lines were peeled off with 0.02% -EDTA Solution (Nacalai Tesque), washed with PBS, and then RPMI1640 medium (GIBCO-BRL) ) And centrifuged at 300 G for 5 minutes to remove the supernatant. After adding the same medium and washing by centrifugation, the cell suspension prepared to an optimal concentration was transplanted subcutaneously at a right arm of SCID mouse female 6-8 weeks old (manufactured by CLEA Japan, Inc.) in an amount of 100 μL. From the same day, the antibody administration group received an antibody solution diluted with PBS, and the control group received 100 μL of PBS alone in the tail vein (5-7 mice per group). Administration was performed twice a week for a total of 8 times, and the tumor diameter was measured with calipers from the time when the tumor was observed. Tumor volume was calculated by the following formula.
(Formula) Tumor volume (mm ³ ) = major axis x minor axis ² x 0.5
The results are shown in FIG. Anti-HB-EGF chimeric antibody KM3966 significantly inhibited tumor growth of ovarian cancer cell lines MCAS and ES-2. Therefore, it was revealed that the anti-HB-EGF chimeric antibody KM3966 has an antitumor effect in an early-stage cancer model. .
(6) -2 Evaluation in advanced cancer model MCAS and ES-2 of human ovarian cancer cell lines and human breast cancer cell line MDA-MB-231 are removed with 0.02% -EDTA Solution (Nacalai Tesque) and washed with PBS. Thereafter, RPMI1640 medium (GIBCO-BRL) was added and centrifuged at 300 G for 5 minutes to remove the supernatant. After adding the same medium and washing by centrifugation, the cell suspension prepared to an optimal concentration was transplanted subcutaneously at a right arm of SCID mouse female 6-8 weeks old (manufactured by CLEA Japan, Inc.) in an amount of 100 μL. Observing the course, mice were selected when the tumor volume reached around 100 mm ^3, and grouped so that the average tumor volume of each group was equal. From the same day, the antibody administration group received an antibody solution diluted with PBS, and the control group received 100 μL of PBS alone in the tail vein (6-7 mice per group). Administration was performed twice a week for a total of 8 times, and the tumor diameter was measured with calipers from the time of antibody administration. Tumor volume was calculated by the following formula.
(Formula) Tumor volume (mm ³ ) = major axis x minor axis ² x 0.5
The results are shown in FIG. As a result, anti-HB-EGF chimeric antibody KM3966 significantly inhibited tumor growth of ovarian cancer cell lines MCAS and ES-2 and breast cancer cell line MDA-MB-231. Therefore, it was revealed that the anti-HB-EGF chimeric antibody KM3966 has antitumor activity in an advanced cancer model.

Evaluation of reactivity and antibody-dependent cytotoxicity (ADCC activity) of anti-HB-EGF antibodies to human blood cancer cell lines (1) HB-EGF expression analysis in human blood cancer cell lines HB-EGF in human blood cancer cell lines In order to evaluate expression, it examined by the fluorescent antibody method. Human acute myeloid leukemia cell lines ML-1 (DSMZ ACC464), MOLM-13 (DSMZ ACC554), MV-4-11 (ATCC CRL9591), HL-60 (ATCC CCL-240), NB-4 (DSMZ ACC207 ), KG-1a (ATCC CCL-246.1) and the human T-cell leukemia cell lines Karpas299 (DSMZ ACC31) and Jurkat (RCB RCB0806) were washed with PBS, adjusted to an optimal concentration, and a 96-well U-bottom plate ( FALCON) was dispensed at 50 μL / well (about 2 × 10 ⁵ cells). 50 μL / well of an anti-HB-EGF mouse antibody KM3566 solution prepared to 20 μg / mL with 1% BSA-PBS was dispensed, stirred with a plate mixer, and allowed to stand on ice for 30 minutes. After washing twice with PBS, 50-fold diluted secondary antibody Anti-mouse Igs / FITC Goat F (ab ') ₂ (DAKO) was added at 50 μL / well, stirred with a plate mixer, protected from light on ice. For 30 minutes. After washing twice with PBS, the fluorescence intensity was measured using a flow cytometer EPICS XL System II v3.0 (manufactured by BECKMAN COULTER). Mouse IgG1 (DAKO) was used as a negative control antibody.

The results are shown in FIG. KM3566 specifically bound to T cell leukemia and acute myeloid leukemia cell lines. Therefore, it was confirmed that HB-EGF is expressed in human blood cancer cell lines.
(2) Antibody-dependent cytotoxic activity (ADCC activity) of anti-HB-EGF chimeric antibody against human blood cancer cell line
The ADCC activity of the HB-EGF chimeric antibody KM3966 against the acute myeloid leukemia cell line in which HB-EGF expression was confirmed was measured according to the following method.
(2) -1 Preparation of target cell solution Human acute myeloid leukemia cell lines ML-1, MOLM-13, MV-4-11, HL-60, NB-4 and KG-1a were washed with PBS, then ADCC After washing with the working medium, the target cell solution was prepared by adjusting to the optimum concentration with the same medium.
(2) -2 Preparation of Effector Cell Solution Peripheral blood mononuclear cells (PBMC) were separated from healthy human peripheral blood by the method described below. 50 mL of healthy human peripheral blood was collected with a syringe containing a small amount of heparin sodium injection N “Shimizu” (manufactured by Shimizu Pharmaceutical Co., Ltd.). The collected peripheral blood was diluted by adding the same amount of physiological saline (manufactured by Otsuka Pharmaceutical) and stirred well. On Polymorphprep (manufactured by NYCOMED) dispensed approximately 6.5 mL into a 15 mL tube (manufactured by Greiner), the same amount of diluted peripheral blood is gently layered, and centrifuged at room temperature for 800 G for 30 minutes to mononuclear cells. The layers were separated. After washing twice with the ADCC medium, it was adjusted to the optimum concentration with the same medium to obtain an effector cell solution.
(2) -3 Measurement of ADCC activity
50 μL of antibody dilution solution is dispensed into each well of a 96-well U-bottom plate (manufactured by FALCON), and 50 μL of the target cell solution prepared in (2) -1 and effector cells prepared in (2) -2 50 μL of the solution was added (the ratio of the effector cell (E) to the target cell (T) was 25), the total amount was 150 μL, and the mixture was reacted at 37 ° C. for 4 hours. Target cell spontaneous release values were obtained by adding 50 μL of target cell solution and 100 μL of medium, and target cell and effector cell spontaneous release values were obtained by adding 50 μL of target cell solution, 50 μL of effector cells and 50 μL of medium. . The target cell total release value was obtained by adding 50 μL of target cell solution and 80 μL of medium, and adding 20 μL of 9% Triton X-100 solution 45 minutes before the end of the reaction. After the reaction, the plate was centrifuged, and the lactate dehydrogenase (LDH) activity in the supernatant was detected by measuring the absorbance using LDH-Cytotoxic Test (manufactured by Wako) according to the attached instructions. ADCC activity was determined by the following formula.
(formula)
ADCC activity (%) = ([absorbance of specimen] − [absorbance of spontaneous release of target cells and effector cells]) / ([absorbance of total release of target cells] − [absorbance of spontaneous release of target cells]) × 100
The results are shown in FIG. The anti-HB-EGF chimeric antibody KM3966 exhibited cytotoxic activity against the HB-EGF-positive human blood cancer cell line in an antibody concentration-dependent manner. Accordingly, the anti-EB-EGF monoclonal antibody and the recombinant antibody of the present invention are not only solid cancers such as ovarian cancer expressing HB-EGF but also blood cancers such as acute myeloid leukemia and T cell leukemia. It was suggested that it may be effective for

Preparation of anti-HB-EGF humanized antibody (1) Design of amino acid sequences of VH and VL of anti-HB-EGF humanized antibody First, the amino acid sequence of VH of anti-HB-EGF humanized antibody was designed as follows. .
The amino acid sequence of FR of human antibody VH for grafting the amino acid sequence of CDR1 to CDR3 of antibody VH represented by SEQ ID NOs: 12 to 14, respectively, was selected. Kabat et al. Classified VHs of various known human antibodies into three subgroups (HSG I-III) based on their amino acid sequence homology, and reported common sequences for each subgroup. [SEQUENCES of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)]. Since these consensus sequences may be less immunogenic in humans, the amino acid sequence of VH of the anti-HB-EGF humanized antibody was designed based on these consensus sequences. In order to produce an anti-HB-EGF humanized antibody with higher binding activity, the design of the anti-HB-EGF mouse antibody KM3566 among the FR amino acid sequences of the consensus sequence of the three subgroups of human antibody VH The FR amino acid sequence having the highest homology with the FR amino acid sequence of VH was selected.

As a result of searching for homology, the homology of HSGI, HSGII and HSGIII was 73.6%, 50.6% and 56.3%, respectively. Therefore, the amino acid sequence of FR in the VH region of KM3566 had the highest homology with subgroup I.
From the above results, the VH CDR amino acid sequence of the anti-HB-EGF mouse antibody KM3566 was transplanted to the appropriate position of the FR amino acid sequence of the consensus sequence of VH subgroup I of the human antibody. However, the 74th Lys in the amino acid sequence of VH of KM3566 described in SEQ ID NO: 9 is not the most frequently used amino acid residue at the corresponding site in the amino acid sequence of human antibody FR mentioned by Kabat et al. Are amino acid residues that are used at a relatively high frequency, and therefore the amino acid residues found in the amino acid sequence of KM3566 described above were used. Thus, the amino acid sequence HV0 of VH of the anti-HB-EGF humanized antibody represented by SEQ ID NO: 22 was designed.

Next, the amino acid sequence of VL of the anti-HB-EGF humanized antibody was designed as follows.
The amino acid sequence of FR of human antibody VL for grafting the amino acid sequence of CDR1 to CDR3 of antibody VL represented by SEQ ID NOs: 15 to 17, respectively, was selected. Kabat et al. Classify VL of various known human antibodies into four subgroups (HSG I to IV) based on their amino acid sequence homology, and further report common sequences for each subgroup. [Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)]. Therefore, as in the case of VH, among the FR amino acid sequences of the consensus sequence of the four subgroups of human antibody VL, the highest homology with the FR amino acid sequence of VL of anti-HB-EGF mouse antibody KM3566. The FR amino acid sequence was selected.

As a result of searching for homology, the homology of HSGI, HSGII, HSGIII and HSGIV was 75.0%, 75.0%, 71.3% and 81.3%, respectively. Therefore, the amino acid sequence of FR of VL of KM3566 had the highest homology with subgroup IV.
Based on the above results, the amino acid sequence of the CDR of the VL of the anti-HB-EGF mouse antibody KM3566 was transplanted to the appropriate position of the FR amino acid sequence of the consensus sequence of subgroup IV of the human antibody VL. However, the 110th Leu in the amino acid sequence of KM3566 VL described in SEQ ID NO: 11 is not the most frequently used amino acid residue at the corresponding site in the amino acid sequence of human antibody FR listed by Kabat et al. Are amino acid residues that are used at a relatively high frequency. Therefore, amino acid residues that are found in the amino acid sequence of KM3566 are used. Thus, the amino acid sequence LV0 of the VL of the anti-HB-EGF humanized antibody represented by SEQ ID NO: 23 was designed.

  The VH amino acid sequence HV0 and VL amino acid sequence LV0 of the anti-HB-EGF humanized antibody designed above are transplanted only with the CDR amino acid sequence of the anti-HB-EGF mouse antibody KM3566 to the FR amino acid sequence of the selected human antibody. It is an array. However, in general, when a humanized antibody is prepared, the binding activity is often lowered only by transplanting the amino acid sequence of the CDR of the mouse antibody to the FR of the human antibody. For this reason, in order to avoid a decrease in binding activity, among amino acid residues of FR that are different between human and mouse antibodies, amino acid residues that are thought to affect binding activity are transferred together with the CDR amino acid sequence transplantation. Have been modified. Therefore, also in this example, FR amino acid residues that are thought to affect the binding activity were identified as follows.

  First, the three-dimensional structure of the antibody V region (HV0LV0) consisting of the VH amino acid sequence HV0 and the VL amino acid sequence LV0 of the anti-HB-EGF humanized antibody designed above was constructed using a computer modeling technique. Use the software AbM (manufactured by Oxford Molecular) for 3D structure coordinate creation, and use the software Pro-Explore (manufactured by Oxford Molecular) or ViewerLite (manufactured by Accelrys) for the display of 3D structures. I went according to the book. A computer model of the three-dimensional structure of the V region of the anti-HB-EGF mouse monoclonal antibody KM3566 was also constructed in the same manner. Furthermore, in the amino acid sequence of VH of HV0LV0 and FR of VL, an amino acid residue that is different from that of anti-HB-EGF mouse antibody KM3566 is selected and modified to the amino acid residue of anti-HB-EGF mouse antibody KM3566 The three-dimensional structural model was constructed in the same manner. The three-dimensional structures of the V regions of these anti-HB-EGF mouse antibodies KM3566 and HV0LV0 and variants were compared, and amino acid residues predicted to affect the antibody binding activity were identified.

As a result, among the amino acid residues of FR of HV0LV0, the three-dimensional structure of the antigen binding site was changed, and as amino acid residues that are thought to affect the binding activity of the antibody, the 9th Ala and 20th amino acid residues in HV0 Val, 30th Thr, 38th Arg, 41st Pro, 48th Met, 67th Arg, 68th Val, 70th Ile, 95th Tyr, and 118th Val, LV0 So we selected 15th Leu, 19th Ala, 21st Ile, 49th Pro, and 84th Leu. Among these selected amino acid residues, at least one or more amino acid sequences were modified to amino acid residues present at the same site of mouse antibody KM3566, and VH and VL of humanized antibodies having various modifications were designed. Specifically, for antibody VH, the 9th Ala of the amino acid sequence represented by SEQ ID NO: 22 is Thr, the 20th Val is Leu, the 30th Thr is Arg, and the 38th Arg is Lys. 41st Pro to Thr, 48th Met to Ile, 67th Arg to Lys, 68th Val to Ala, 70th Ile to Leu, 95th Tyr to Phe, At least one of the amino acid modifications replacing Leu with 118th Val was introduced. For VL, the 15th Leu of the amino acid sequence represented by SEQ ID NO: 23 is Val, the 19th Ala is Val, the 21st Ile is Met, the 49th Pro is Ser, and 84 At least one modification was introduced among the amino acid modifications replacing the second Leu with Val.
(2) Construction of cDNA encoding VH of anti-HB-EGF humanized antibody cDNA encoding the amino acid sequence HV0 of VH of anti-HB-EGF humanized antibody designed in this Example (1) was obtained using PCR. It was constructed as follows.

  First, the designed amino acid sequence was linked to the H chain secretory signal sequence of the anti-HB-EGF mouse antibody KM3566 represented by the 1st to 19th positions of SEQ ID NO: 9 to obtain a complete antibody amino acid sequence. Next, the amino acid sequence was converted into a gene codon. When multiple gene codons exist for one amino acid residue, consider the frequency of use found in the base sequence of the antibody gene [SEQUENCES of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)] And the corresponding gene codons were determined. By linking the determined gene codons, the base sequence of the cDNA encoding the amino acid sequence of the complete antibody V region was designed, and further, the base sequence of the primer for amplification during PCR reaction (humanized) at the 5 'end and 3' end Including a restriction enzyme recognition sequence for cloning into an antibody expression vector). Divide the designed base sequence into 4 base sequences in total of about 100 bases from the 5 ′ end side (adjacent base sequences should have an overlapping sequence of about 20 bases at their ends), Synthetic DNA (SEQ ID NOs: 24-27) was synthesized in the order of alternating antisense strands.

  Each synthetic DNA (SEQ ID NO: 24-27) is added to 50 μL of the reaction solution so that the final concentration is 0.1 μmol / L, and 0.5 μmol / L T3 primer (Takara Shuzo), 0.5 μmol / L T7 primer ( Using Takara Shuzo) and 1 unit of KOD polymerase (Toyobo Co., Ltd.), PCR was performed according to the instruction manual attached to KOD polymerase. The reaction conditions in this case were in accordance with the conditions described in the instruction manual (cycles of 94 ° C. for 30 seconds, 50 ° C. for 30 seconds, 74 ° C. for 60 seconds for 30 cycles). The reaction solution was precipitated with ethanol, dissolved in sterilized water, treated with an appropriate restriction enzyme, and then ligated to a plasmid pBluescript II SK (-) (Stratagene). Using the recombinant plasmid DNA solution thus obtained, E. coli DH5α strain was transformed, plasmid DNA was prepared from the transformed strain, and BigDye Terminator Cycle Sequencing FS Ready Reaction Kit (Applied Biosystems) was prepared. As a result of analyzing the base sequence, a plasmid having the target base sequence was obtained.

Next, the modification of the FR amino acid residues designed in this Example (1) is carried out by preparing a synthetic DNA having a mutation and performing the above PCR or plasmid DNA containing the cDNA encoding HV0 prepared above. PCR was performed using a synthetic DNA having a mutation as a primer, and the amplified gene fragment was isolated. The modified gene codon of the amino acid residue was made to be the gene codon found in the anti-HB-EGF mouse antibody KM3566. In addition, unless otherwise specified, a cycle of 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 72 ° C. for 60 seconds was allowed to react in 35 cycles of PCR reaction. PCR reaction was performed using KOD-plus polymerase (TOYOBO). The synthetic DNA used is manufactured by Fasmac.
(3) Construction of cDNA encoding VL of anti-HB-EGF humanized antibody The cDNA encoding the amino acid sequence of VL of the anti-HB-EGF humanized antibody designed in this Example (1) was obtained using PCR. It was built as follows.

  First, the designed amino acid sequence was linked to the secretory signal sequence of the L chain of the anti-HB-EGF mouse antibody KM3566 shown in the 1st to 20th positions of SEQ ID NO: 11 to obtain a complete antibody amino acid sequence. Next, the amino acid sequence was converted into a gene codon. When multiple gene codons exist for a single amino acid residue, consider the frequency of use [SEQUENCES of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)] found in the base sequence of the antibody gene And the corresponding gene codons were determined. Connect the determined gene codons to design the base sequence of the cDNA that encodes the amino acid sequence of the complete antibody V region. Furthermore, the 5 'and 3' ends are the base sequences for the amplification primers used in the PCR reaction (humanized) Including a restriction enzyme recognition sequence for cloning into an antibody expression vector). Divide the designed base sequence into 4 base sequences of about 100 bases each from the 5 'end side (adjacent base sequences should have an overlapping sequence of about 20 bases at their ends) Synthetic DNA (SEQ ID NOs: 28-31) was synthesized in the order of alternating antisense strands.

  Each synthetic DNA (SEQ ID NO: 28 to 31) is added to 50 μL of the reaction solution so that the final concentration is 0.1 μmol / L, and 0.5 μmol / L T3 primer (Takara Shuzo), 0.5 μmol / L T7 primer ( Using Takara Shuzo) and 1 unit of KOD polymerase (Toyobo Co., Ltd.), PCR was performed in the same manner as in (2) above according to the instruction manual attached to KOD polymerase. The reaction solution was precipitated with ethanol, dissolved in sterilized water, treated with an appropriate restriction enzyme, and then ligated to the plasmid pBluescript II SK (-) (Stratagene). Using the recombinant plasmid DNA solution thus obtained, E. coli DH5α strain was transformed, plasmid DNA was prepared from the transformed strain, and BigDye Terminator Cycle Sequencing FS Ready Reaction Kit (Applied Biosystems) was used. As a result of analyzing the base sequence, plasmid pBS / LV0 having the target base sequence was obtained.

  Next, the modification of the FR amino acid residue designed in this Example (1) is carried out by preparing a synthetic DNA having a mutation and performing the above PCR or the plasmid DNA containing the cDNA encoding LV0 prepared above. PCR was performed using a synthetic DNA having a mutation as a primer and isolation of the amplified gene fragment. The modified gene codon of the amino acid residue was made to be the gene codon found in the anti-HB-EGF mouse antibody KM3566.

Unless otherwise specified, PCR reaction was performed at 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 72 ° C. for 60 seconds in 35 cycles using KOD-plus polymerase (manufactured by TOYOBO). The synthetic DNA used was manufactured by Fasmac.
(4) Construction of anti-HB-EGF humanized antibody expression vector
The respective cDNAs encoding HV0 and LV0 obtained in Examples (2) and (3) are encoded at appropriate positions of the humanized antibody expression vector pKANTEX93 described in WO97 / 10354, or variants thereof. cDNAs were inserted to construct various anti-HB-EGF humanized antibody expression vectors.
(5) Stable expression using anti-HB-EGF humanized antibody using animal cells and acquisition of purified antibody Stable expression using anti-HB-EGF humanized antibody using animal cells and purification of the antibody from the culture supernatant were performed. Performed in a manner similar to that described in Example 6 (2) and (3).

Analysis of binding epitope of anti-HB-EGF antibody The following analysis was performed on the binding epitope of anti-HB-EGF antibodies KM3566, KM3579, and KM3966 against human HB-EGF.
(1) Construction of mutant human HB-EGF full-length gene-transfected cells
Anti-HB-EGF antibodies KM3566, KM3579, and KM3966 all react with human HB-EGF and do not show cross-reactivity with mouse HB-EGF. Therefore, in the amino acid sequence of the EGF-like domain of human HB-EGF, 10 mutant human HB-EGF full-length proteins in which 10 amino acids different from mouse HB-EGF are replaced with mouse-derived amino acids one by one. Gene-transfected cells that express (hereinafter referred to as mutant HB-EGF) were constructed, and the binding epitopes were analyzed by measuring the binding activity of the anti-HB-EGF antibody to these cells. The 10 types of mutant HB-EGF prepared are shown below.
(1) Mutant HB-EGF in which the 115th phenylalanine from the N-terminus is replaced with tyrosine (hereinafter referred to as F115Y)
(2) Mutant HB-EGF in which 122th lysine from the N-terminus is replaced with arginine (hereinafter referred to as K122R)
(3) Mutant HB-EGF in which 124th valine from N-terminal is replaced with leucine (hereinafter referred to as V124L)
(4) Mutant HB-EGF in which the 125th lysine from the N-terminus is replaced with glutamine (hereinafter referred to as K125Q)
(5) Mutant HB-EGF in which the leucine at position 127 from the N-terminus is substituted with phenylalanine (hereinafter referred to as L127F)
(6) Mutant HB-EGF in which the 129th alanine from the N-terminus is replaced with threonine (hereinafter referred to as A129T)
(7) Mutant HB-EGF obtained by substituting 133th isoleucine from the N-terminus with lysine (hereinafter referred to as I133K)
(8) Mutant HB-EGF in which histidine at position 135 from the N-terminus is replaced with leucine (hereinafter referred to as H135L)
(9) Mutant HB-EGF in which glutamic acid at position 141 from the N-terminus is replaced with histidine (hereinafter referred to as E141H)
(10) Mutant HB-EGF in which the 147th serine from the N-terminus is replaced with threonine (hereinafter referred to as S147T).

Furthermore, the following human / mouse chimeric HB-EGF full-length gene-transferred cells were constructed as a positive control.
(11) A human / mouse chimeric HB- in which the first to 49th sequences from the N-terminus are mouse HB-EGF-derived sequences, and the 50th to 208th sequences from the N-terminus are human HB-EGF-derived sequences. EGF (hereinafter referred to as pRTHGC-6) was prepared. Since all EGF-like domains are human HB-EGF-derived sequences, they were used as positive controls.

  The above mutant HB-EGF and human / mouse chimeric HB-EGF transient expression plasmids were prepared using the method of Mekada et al. (J. Bio. Chem., Vol. 272, 27084-27090, 1997). did. Mouse LMTK-cells (ATCC CCL-1.3) were cultured in Dulbecco's modified Eagle's medium supplemented with 100 unit / mL penicillin G, 100 μg / mL streptomycin, and 10% fetal bovine serum. Each of the above expression plasmids was introduced into mouse LMTK-cells by the calcium phosphate method and then cultured for 48 hours, and used in the subsequent experiments.

As a negative control, cells in which only the vector was introduced into mouse LMTK-cells (hereinafter referred to as mock) were used.
(2) Analysis of binding activity of anti-HB-EGF antibody using mutant HB-EGF gene-introduced cells First, 1 × 10 ⁵ mutant HB-EGF gene-introduced cells, human / mouse chimeric HB-EGF gene-introduced cells, And mock, biotinylated anti-HB-EGF antibody diluted to 2 μg / mL with binding buffer (Ham's F12 supplemented with non-essential amino acids, 20 mM Hepes-NaOH (pH 7.2), 10% fetal bovine serum) (KM3566, KM3579, KM3966) were reacted at 4 ° C. for 2 hours. After the reaction, it was washed _twice with ice-cold washing buffer (PBS supplemented with 0.5 mM CaCl ₂ , 0.5 mM MgCl ₂ , 0.1% fetal bovine serum), and then once with PBS (+) (0.5 mM in PBS). Washed with CaCl ₂ and 0.5 mM MgCl ₂ . A formaldehyde solution diluted to 1.8% with PBS (+) was added to the washed cells, and the cells were fixed at 4 ° C. for 20 minutes. Next, after washing once with PBS (+), it was treated with glycine solution (0.2 M-glycine, 100 mM Tris, pH 8.1) for 4 C for 20 minutes, and then incubated with washing buffer at 4 ° C. for 20 minutes. Next, HRP-conjugated streptavidin diluted to 0.1 μg / mL with a binding buffer was reacted at 4 ° C. for 1 hour, washed twice with a washing buffer, and washed twice with PBS (+). Color was developed using a peroxidase detection kit (Nacalai Tesque, ELISA POD substrate OPD kit), the absorbance at 492 nm was measured, and the HRP activity bound to the cells was measured.

The value obtained by subtracting the absorbance for mock from the absorbance for various mutant HB-EGF gene-introduced cells of anti-HB-EGF antibody and human / mouse chimeric HB-EGF gene-introduced cells was defined as A value.
Next, in order to analyze the expression level of the mutant HB-EGF protein expressed on the cell membrane of mouse LMTK-cells, an anti-HB-EGF rabbit polyclonal antibody (antibody name; H-6) that binds equally to all mutant HB-EGFs. , An antibody prepared by immunizing rabbits with a synthetic peptide of human HB-EGF from the N-terminal to the 54th to 73rd cross-linked to Sepharose CL-6B, EMBO J., 13, 2322-2330 , 1994), the absorbance of the mutant HB-EGF gene-introduced cells, human / mouse chimeric HB-EGF gene-introduced cells, and mock was measured by the same method as described above. However, biotin-labeled H-6 antibody was used at an antibody concentration of 10 μg / mL. The value obtained by subtracting the absorbance for mock from the absorbance for each mutant HB-EGF gene-introduced cell and human / mouse chimeric HB-EGF gene-introduced cell of H-6 antibody was defined as B value.

In order to correct the difference in the expression level of the transfected cells, the A / B value was determined by dividing the A value by the B value. The ratio of the A / B value to various mutant HB-EGFs when the A / B value for the positive control pRTHGC-6 of the anti-HB-EGF antibody was taken as 100% was calculated and this was relative to the various mutant HB-EGFs. Binding activity.
The results are shown in FIG. The anti-HB-EGF monoclonal antibody KM3566 hardly bound to I133K, H135L, and S147T compared to pRTHGC-6. Therefore, it was revealed that the anti-HB-EGF monoclonal antibody KM3566 recognizes an epitope containing the 133rd I, 135th H and 147th S amino acids. Similarly to the anti-HB-EGF monoclonal antibody KM3566, the anti-HB-EGF chimeric antibody KM3966 having the same antibody variable region also hardly binds to I133K and H135L compared to pRTHGC-6, and S147T Then, the binding activity decreased to about 1/3. Therefore, the anti-HB-EGF chimeric antibody KM3966, like the anti-HB-EGF monoclonal antibody KM3566, clearly recognizes an epitope containing the 133rd I, 135th H and 147th S amino acids. became.

The anti-HB-EGF monoclonal antibody KM3579 did not bind to E141H alone compared to pRTHGC-6, and all other mutant HB-EGFs showed binding activity equivalent to pRTHGC-6. Therefore, it was revealed that the anti-HB-EGF monoclonal antibody KM3579 recognizes an epitope containing the 141st E amino acid.
The above results revealed that anti-HB-EGF monoclonal antibody KM3566 and anti-HB-EGF chimeric antibody KM3966 and anti-HB-EGF monoclonal antibody KM3579 recognize different epitopes of HB-EGF.

The present invention provides monoclonal antibodies and antibody fragments thereof that bind to HB-EGF, membrane-type HB-EGF, and secretory HB-EGF that are bound to cell membranes.

Sequence Listing Free Text SEQ ID NO: 18—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 19—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 20—Description of artificial sequence: synthetic DNA
SEQ ID NO: 21—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 22-description of artificial sequence: amino acid sequence of humanized antibody SEQ ID NO: 23-description of artificial sequence: amino acid sequence of humanized antibody SEQ ID NO: 24-description of artificial sequence: synthetic DNA
SEQ ID NO: 25—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 26—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 27—Description of artificial sequence: synthetic DNA
SEQ ID NO: 28—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 29—Description of artificial sequence: synthetic DNA
SEQ ID NO: 30—Description of artificial sequence: synthetic DNA
SEQ ID NO: 31—Description of artificial sequence: synthetic DNA

Claims (24)

Monoclonal binding to heparin binding epidermal growth factor-like growth factor (hereinafter referred to as HB-EGF), membrane-type HB-EGF and secretory HB-EGF. An antibody or antibody fragment thereof. The monoclonal antibody or antibody fragment thereof according to claim 1, which binds to an epidermal growth factor-like domain (EGF-like domain) of HB-EGF, membrane-type HB-EGF and secretory HB-EGF bound to a cell membrane. 3. The monoclonal antibody or antibody fragment thereof according to claim 1 or 2, which inhibits binding between secretory HB-EGF and HB-EGF receptor. The monoclonal antibody or antibody fragment thereof according to any one of claims 1 to 3, which has neutralizing activity against secretory HB-EGF. The monoclonal antibody or antibody fragment thereof according to any one of claims 1 to 4, which binds to a binding region between secretory HB-EGF and an HB-EGF receptor or diphtheria toxin. 6. The monoclonal antibody or the antibody fragment thereof according to claim 1, which binds to an epitope comprising at least one amino acid among the 133rd, 135th, and 147th amino acid sequences represented by SEQ ID NO: 2. 7. The monoclonal antibody or antibody fragment thereof according to claim 6, which binds to an epitope comprising amino acids 133, 135 and 147 of the amino acid sequence represented by SEQ ID NO: 2. 6. The monoclonal antibody or antibody fragment thereof according to claim 1, which binds to an epitope comprising the 141st amino acid of the amino acid sequence represented by SEQ ID NO: 2. The monoclonal antibody or antibody fragment thereof according to any one of claims 1 to 3, 5 and 8, which binds to an epitope to which a monoclonal antibody produced by hybridoma KM3579 (FERM BP-10491) binds. The monoclonal antibody or antibody fragment thereof according to any one of claims 1 to 7, which binds to an epitope to which a monoclonal antibody produced by hybridoma KM3567 (FERM BP-10573) binds. The monoclonal antibody or antibody fragment thereof according to any one of claims 1 to 7, which binds to an epitope to which a monoclonal antibody produced by hybridoma KM3566 (FERM BP-10490) binds. Complementarity determining region (hereinafter referred to as CDR) 1, CDR2 and CDR3 of the antibody heavy chain variable region (hereinafter referred to as VH) are the amino acid sequences represented by SEQ ID NOs: 12, 13 and 14, respectively. And the CDR1, CDR2, and CDR3 of the light chain variable region (hereinafter referred to as VL) of the antibody comprise the amino acid sequences represented by SEQ ID NOs: 15, 16, and 17, respectively. The monoclonal antibody or antibody fragment thereof according to any one of the above. The antibody or antibody fragment thereof according to any one of claims 1 to 12, wherein the monoclonal antibody is a gene recombinant antibody. 14. The antibody or antibody fragment thereof according to claim 13, wherein the recombinant antibody is selected from a human chimeric antibody, a humanized antibody and a human antibody. 15. The human chimeric antibody or the antibody thereof according to claim 14, wherein VH of the human chimeric antibody comprises the amino acid sequence represented by SEQ ID NO: 9, and VL comprises the amino acid sequence represented by SEQ ID NO: 11. fragment. The VH of the humanized antibody is the amino acid sequence represented by SEQ ID NO: 22 or the 9th Ala of the amino acid sequence represented by SEQ ID NO: 22 to Thr, the 20th Val to Leu, and the 30th Thr to Arg , 38th Arg to Lys, 41st Pro to Thr, 48th Met to Ile, 67th Arg to Lys, 68th Val to Ala, 70th Ile to Leu, 95 An amino acid sequence into which at least one modification selected from a modification in which the thirth Tyr is replaced with Phe and the 118th Val is replaced with Leu, and
The VL of the humanized antibody is the amino acid sequence represented by SEQ ID NO: 23 or the 15th Leu of the amino acid sequence represented by SEQ ID NO: 23 to Val, the 19th Ala to Val, and the 21st Ile to Met. 15. The humanized antibody or antibody fragment thereof according to claim 14, comprising an amino acid sequence into which at least one modification selected from a modification in which 49th Pro is replaced with Ser and 84th Leu is replaced with Val. The antibody fragment is selected from peptides including Fab, Fab ′, F (ab ′) ₂ , single chain antibody (scFv), dimerized V region (Diabody), disulfide stabilized V region (dsFv) and CDR. The antibody fragment according to any one of claims 1 to 16, which is an antibody fragment. DNA encoding the antibody or antibody fragment thereof according to any one of claims 1 to 17. A recombinant vector containing the DNA according to claim 18. A transformant obtained by introducing the recombinant vector according to claim 19 into a host cell. A transformant according to claim 20 is cultured in a medium, and the antibody or antibody fragment thereof according to any one of claims 1 to 17 is produced and accumulated in the culture, and the antibody or the antibody is cultured from the culture. The method for producing an antibody or antibody fragment thereof according to any one of claims 1 to 17, wherein the fragment is collected. A medicament comprising the antibody or antibody fragment thereof according to any one of claims 1 to 17 as an active ingredient. A therapeutic agent for a disease involving HB-EGF, comprising the antibody or antibody fragment thereof according to any one of claims 1 to 17 as an active ingredient. 24. The therapeutic agent according to claim 23, wherein the disease involving HB-EGF is cancer.